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Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization
GENOMICS
7, 127-130
(1990)
SHORT COMMUNICATION Rapid Analysis of Mouse-Hamster Hybrid Cell Lines by in Situ Hybridization ANN
L. BOYLE,* PETER kHTER,t
AND DAVID
C. WARD*‘t
Departments of “Molecular Biophysics and Biochemistry and ti-fuman Genetics, Yale University School of Medicine, New Haven, Connecticut 0657 0 Received
November
banding combined with Hoechst 33258 staining of mouse heterochromatin has been used (Kozak et al., 1977). However, quantitative evaluation of banded chromosomes in mouse-hamster hybrid cells requires highly skilled personnel and is quite time consuming. Moreover, detection of small rearranged chromosome pieces, such as translocations and insertions, may be extremely difficult. More recently, human-rodent hybrid cell lines have been rapidly analyzed by in situ hybridization using biotinylated total genomic human DNA as the probe (Schardin et al., 1985; Manuelidis, 1985; Durnam et al., 1985; Pinkel et al., 1986). The human chromosome component of the metaphase spreads was clearly seen against the rodent background by fluorescent or calorimetric detection methods. The technique permits delineation of human chromosomes in interphase as well as metaphase and takes advantage of the species-specific nature of the repetitive DNA elements in the human and rodent genomes. Experiments were designed to determine whether the repetitive sequences of mouse and hamster were sufficiently divergent to allow in situ hybridization analysis with genomic DNA. For this analysis we chose the cell line MAE28, a mouse-hamster hybrid cell line containing an X1’ fusion chromosome in approximately 50% of the cells (Pravtcheva et al, 1981). Initially, we anticipated possible cross-hybridization of the mouse repetitive sequencesto hamster DNA, which would result in background staining of the parental genome. By adding total genomic DNA to the probe and allowing the denatured DNA mixture to partially reanneal before applying it to the sample slide, we hoped to eliminate this unwanted signal, as was done in previous in situ suppression hybridization experiments using human DNA (Landegent et al., 1987; Lichter et al., 1988; Cremer et al., 1988; Pinkel et al, 1988). Biotinylated mouse DNA (5-40 Fg/ml) was hybridized to slides containing metaphase spreadsfrom MAE28 cells,
In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome Xl’. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line. o ISDO Academic
Press.
8, 1989
Inc.
The use of human-rodent and mouse-hamster somatic cell hybrid lines has greatly enhanced efforts to map both human and mouse genes. The accuracy of the chromosome assignments relies on a correct and reliable analysis of the chromosome content of the hybrid lines used. Very often, these cell lines are unstable with respect to chromosomal content and arrangement, and therefore the lines must be reexamined periodically. Analysis has been accomplished primarily by employing banding and differential staining techniques. For example, the alkaline Giemsa (Gil) protocol is used to differentiate species-specific chromosomes on the basis of color in human-rodent lines (Bobrow and Cross, 1974). In mouse-hamster hybrid cell lines, the preferential staining of mouse centromeres by Hoechst 33258 can be used to differentiate mouse from hamster chromosomes (Hilwig and Gropp, 1972). This method, however, cannot detect many structural changes and interspecies chimeric chromosomes. To partly circumvent these problems, Giemsa 127
All
Copyright Q 1990 rights of reproduction
OSSS-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
128
SHORT
COMMUNICATION
SHORT
COMMUNICATION
with and without 200 pg/ml of hamster DNA as competitor (and 800 pg/ml of salmon sperm DNA). Both the suppressed and the unsuppressed hybridization conditions gave equally strong signals for the mouse chromosomes, with virtually no background on the parental hamster chromosomes. Thus, there is enough sequence diversity between rodent species to exploit this technique to analyze mouse-hamster hybrid lines without suppression. Analysis of the results was fast and easy and confirmed the presence of the fusion chromosome in approximately half of the cells examined. In one particular subculture of MAE28 however, multiple chromosomal rearrangements occurred and the mouse DNA manifested itself in a variety of patterns. Figure 1A depicts a spread with the expected single Xl2 fusion chromosome. In addition, interspecies chromosomal rearrangements such as translocations (Fig. 1B) and insertions (Fig. 1C) were readily detected. The metaphase cell in Fig. 1D contains no mouse chromosomes but does contain mouse DNA in the form of minutes. Finally, growth in selective HAT medium sometimes results in the amplification of the Xl2 mouse fusion chromosome as seen in Fig. 1E. This subculture of MAE28 must have been subjected to stressful or nonoptimal culture conditions, thereby destabilizing the mouse chromosome component. As an alternative approach, as well as a way of generating mouse chromosome-specific probes, we also performed the reciprocal experiment whereby total DNA isolated from several hybrid lines was used on normal female mouse metaphase spreads. Biotinylated DNA from the cell line MalOb (Terao et al., 1988), also containing mouse chromosomes X and 12, clearly decorates the two mouse chromosome pairs in both metaphase (Fig. 1F) and interphase. The signal is not distributed uniformly over the chromosome length, reflecting either an underrepresentation of some sequences or the representation of certain repetitive DNA in the hybrid DNA. Similar chromosome-specific labeling of chromosomes X and 12 was achieved with MAE28 DNA; however, in this case, the centromeres of the other mouse chromosomes were also stained. This may be due to nonoptimal suppression conditions. DNA from cell line MAE19 (Pravtcheva et al., 1981),
129
which contains chromosomes X and 1, also specifically labeled the two corresponding murine chromosome pairs (not shown). The use of high probe concentrations is necessary because of the low percentage of mouse sequences relative to the complete hamster complement in the hybrid cell. The amount of probe DNA required is also influenced by the percentage of cells that contain mouse DNA, and optimal concentrations must be determined empirically for each cell line. At these higher probe concentrations, it becomes necessary to suppress both the hamster and the mouse highly repetitive sequences. For some cell line subcultures that presumably have a very low percentage of cells with mouse chromosomes, chromosome-specific signals are absent but centromere and telomere labeling of chromosomes is observed (data not shown). The telomere labeling is due to unsuppressed hamster telomere repeat sequences, as such results are also obtained when one hybridizes similar concentrations of biotinylated hamster DNA to mouse spreads (Fig. 1G). Furthermore, the signal pattern shown in Fig. 1G is similar to that published for the conserved telomeric repeat sequence (TTAGGG), (Moyzis et al., 1988). This method of chromosome analysis is rapid and the results are easily scored by workers not trained in the subtleties of chromosomal banding. The procedure offers the additional advantage that the mouse component of the cells is identified regardless of the quality of the hybrid line metaphase spreads. Accurate analysis of banding patterns, especially if only fragments of chromosomes are present, relies heavily on well-spread, extended chromosomes of good morphology. A number of different highly repetitive mouse sequences have been hybridized to mouse metaphase spreads to produce distinct banding patterns (A. Boyle and D. Ward, unpublished results). These probes can be used to obtain a banding pattern on only those parts of the chromosomes that are of murine origin. One repeat, which produces a Giemsa-like banding pattern, was hybridized to MAE28 metaphase spreads. (Characterization of this probe will be published elsewhere.) The single mouse chromosome displays a banding pattern identical to that published for the Xl2 fusion chromosome of MAE28 (Pravtcheva et al., 1981) (see Fig.
FIG. 1. (A-E) Biotinylated mouse DNA from cell line Cl27 (8) (5 pg/ml) was hybridized without preannealing with competitor DNA (7) to MAE28 metaphase spreads prepared by standard methods (17). Murine DNA was detected using fluorescein-labeled avidin (green) and the chromosomes were counterstained with propidium iodide (red). (A) The X’r fusion chromosome. (B and C) Interspecies chromosomes exhibiting translocations and insertions, respectively. (D) Minutes containing mouse DNA. The insert shows the same spread with propidium iodide alone, indicating that the signal is DNA and not nonspecific background fluorescence. (E) Amplification of the mouse chromosome. (F-H) Female mouse metaphase spreads from spleen cells were prepared according to Sawyer et al. (14) using 6 ag/ml of concanavalin A and 150 pg/ml of 2-mercaptoethanol for cell stimulation. After 48 h, ethidium bromide (final concentration of 25 &4) and 0.1 &ml of colcemid were added for 30 min. (F) Suppression hybridization was performed using 200 pg/ml of biotinylated MalOb DNA as probe, and both 250 pg/ml of E36 hamster cell line DNA (13) and 200 pg/ml of Cl27 mouse DNA as competitor DNA. Experimental details are described by Lichter et al. (7). (G) Biotinylated E36 hamster DNA (1000 pg/ml) (suppressed with 400 pg/ml of Cl27 mouse DNA and 600 pg/ml of E36 hamster DNA) hybridized to a mouse metaphase spread. (H) Biotinylated mouse repetitive probe (8 pg/ml) hybridized to MAE28 metaphase spread Arrowhead and arrow indicate portions of the fusion chromosome that are derived from chromosomes X and 12, respectively.
130
SHORT
COMMUNICATION
1H). Such hybridization banding methods should prove useful if chromosome identification is required. The use of biotinylated hybrid DNA to decorate specific mouse chromosomes provides an alternative approach to hybrid line analysis. In this case, however, various insertions and rearrangements are not observed and an assessment is based on the extent of labeling of the targeted chromosomes. Nevertheless, appropriate cell lines do provide a valuable source of mouse chromosome-specific probes, as mouse flow-sorted chromosome libraries, unlike human chromosomespecific libraries, are not yet available. Thus, under appropriate suppression conditions, hybrid DNA can be used both to label entire metaphase chromosomes and to delineate interphase chromosome domains, which should also prove useful for topography studies.
interspecific mouse/hamster 105: 169-117.
7.
a.
1.
BOBROW, M., AND CROSS, J. (1974). Differential human and mouse chromosomes in interspecific Nature (London) 251: 77-79.
staining of cell hybrids.
10.
11.
12.
14.
2. CREMER,
T., LICHTER, P., BORDEN, J., WARD, D. C., AND MANUELIDIS, L. (1988). Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum. Genet. 80: 235-
246. 3. DURNAM,
D. M., GELINAS, R. E., AND MYERSON, D. (1985). Detection of species specific chromosomes in somatic cell hybrids. Somatic Cell Mol. Genet. 11: 571-577.
4. HILWIG,
I., AND GROPP, A. (1972). Staining erochromatin in mammalian chromosomes chrome. Exp. Cell Res. 76: 122-126.
5. KOZAK, sequential
of constitutive hetwith a new fluoro-
C. A., LAWRENCE, J. B., AND RUDDLE, F. H. (1977). staining technique for the chromosomal analysis
A of
Exp. Cell Res.
J. E., JANSEN IN DE WAL, N., DIRKS, R. W., BAAS, F., AND VAN DER PLOEG, M. (1987). Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370. LICHTER, P., CREMER, T., BORDEN, J., MANUELIDIS, L., AND WARD, D. C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-234. Lowy, D. R., RANDS, E., AND SCOLNICK, E. M. (1979). Helperindependent transformation by unintegrated Harvey sarcoma virus DNA. J. Viral. 26: 291-299.
9. MANUELIDIS,
13.
REFERENCES
cell hybrids.
6. LANDEGENT,
ACKNOWLEDGMENTS We thank Dr. Dimitrina Pravtcheva and Dr. Frank Ruddle for generously providing us with DNA from the cell lines MalOb and MAE19 and for the E36 cell line. Dr. Arthur Horwich generously supplied the MAE28 cell cultures. P.L. was supported by a stipend from the Deutsche Forschungsgemeinschaft. The research was funded by Grants GM-40115 and GM-41596 from the National Institutes of Health.
somatic
L. (1985). Individual interphase chromosome domains revealed by in situ hybridization. Hum. Genet. 71: 288293. MOYZIS, R. K., BUCKINGHAM, J. M., CRAM, L. S., DANI, M., DEAVEN, L. L., JONES, M. D., MEYNE, J., RATLIFF, R. L., AND WU, J. (1988). A highly conserved repetitive DNA sequence, (TTAGGG),, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. USA 86: 6622-6626. PINKEL, D., STRAUME, T., AND GRAY, J. W. (1986). Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934-2936. PINKEL, D., LANDEGENT, J., COLLINS, C., FLJSCOE, J., SEGRAVES, R., LUCAS, J., AND GRAY, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 86: 9138-9142. PRAVTCHEVA, D. D., DELEO, A. B., RUDDLE, F. H., AND OLD, L. J. (1981). Chromosome assignment of the tumor-specific antigen of a 3-methylcholanthrene-induced mouse sarcoma. J. Exp. Med. 154: 964-977. SAWYER, J. R., MOORE, M. M., AND HOZJER, J. C. (1987). High resolution G-banded chromosomes of the mouse. Chromosomu
95:350-358. 15.
16.
17.
SCHARDIN, M., CREMER, T., HAGER, H. D., AND LANG, M. (1985). Specific staining of human chromosomes in Chinese hamster X man hybrid cell lines demonstrates interphase chromosome territories. Hum. Genet. 71: 281-287. TERAO, M., PRAVTCHEVA, D., RUDDLE, F., AND MINTZ, B. (1988). Mapping of gene encoding mouse placental alkaline phosphatase to chromosome 4. Somatic Cell Mol. Genet. 14: 211-215. WAHL, G. M., Vrrro, L., PADGETT, R. A., AND STARK, G. R. (1982). Single copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. Mol. Cell. Biol. 2: 308-319.
7, 127-130
(1990)
SHORT COMMUNICATION Rapid Analysis of Mouse-Hamster Hybrid Cell Lines by in Situ Hybridization ANN
L. BOYLE,* PETER kHTER,t
AND DAVID
C. WARD*‘t
Departments of “Molecular Biophysics and Biochemistry and ti-fuman Genetics, Yale University School of Medicine, New Haven, Connecticut 0657 0 Received
November
banding combined with Hoechst 33258 staining of mouse heterochromatin has been used (Kozak et al., 1977). However, quantitative evaluation of banded chromosomes in mouse-hamster hybrid cells requires highly skilled personnel and is quite time consuming. Moreover, detection of small rearranged chromosome pieces, such as translocations and insertions, may be extremely difficult. More recently, human-rodent hybrid cell lines have been rapidly analyzed by in situ hybridization using biotinylated total genomic human DNA as the probe (Schardin et al., 1985; Manuelidis, 1985; Durnam et al., 1985; Pinkel et al., 1986). The human chromosome component of the metaphase spreads was clearly seen against the rodent background by fluorescent or calorimetric detection methods. The technique permits delineation of human chromosomes in interphase as well as metaphase and takes advantage of the species-specific nature of the repetitive DNA elements in the human and rodent genomes. Experiments were designed to determine whether the repetitive sequences of mouse and hamster were sufficiently divergent to allow in situ hybridization analysis with genomic DNA. For this analysis we chose the cell line MAE28, a mouse-hamster hybrid cell line containing an X1’ fusion chromosome in approximately 50% of the cells (Pravtcheva et al, 1981). Initially, we anticipated possible cross-hybridization of the mouse repetitive sequencesto hamster DNA, which would result in background staining of the parental genome. By adding total genomic DNA to the probe and allowing the denatured DNA mixture to partially reanneal before applying it to the sample slide, we hoped to eliminate this unwanted signal, as was done in previous in situ suppression hybridization experiments using human DNA (Landegent et al., 1987; Lichter et al., 1988; Cremer et al., 1988; Pinkel et al, 1988). Biotinylated mouse DNA (5-40 Fg/ml) was hybridized to slides containing metaphase spreadsfrom MAE28 cells,
In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome Xl’. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line. o ISDO Academic
Press.
8, 1989
Inc.
The use of human-rodent and mouse-hamster somatic cell hybrid lines has greatly enhanced efforts to map both human and mouse genes. The accuracy of the chromosome assignments relies on a correct and reliable analysis of the chromosome content of the hybrid lines used. Very often, these cell lines are unstable with respect to chromosomal content and arrangement, and therefore the lines must be reexamined periodically. Analysis has been accomplished primarily by employing banding and differential staining techniques. For example, the alkaline Giemsa (Gil) protocol is used to differentiate species-specific chromosomes on the basis of color in human-rodent lines (Bobrow and Cross, 1974). In mouse-hamster hybrid cell lines, the preferential staining of mouse centromeres by Hoechst 33258 can be used to differentiate mouse from hamster chromosomes (Hilwig and Gropp, 1972). This method, however, cannot detect many structural changes and interspecies chimeric chromosomes. To partly circumvent these problems, Giemsa 127
All
Copyright Q 1990 rights of reproduction
OSSS-7543/90 $3.00 by Academic Press, Inc. in any form reserved.
128
SHORT
COMMUNICATION
SHORT
COMMUNICATION
with and without 200 pg/ml of hamster DNA as competitor (and 800 pg/ml of salmon sperm DNA). Both the suppressed and the unsuppressed hybridization conditions gave equally strong signals for the mouse chromosomes, with virtually no background on the parental hamster chromosomes. Thus, there is enough sequence diversity between rodent species to exploit this technique to analyze mouse-hamster hybrid lines without suppression. Analysis of the results was fast and easy and confirmed the presence of the fusion chromosome in approximately half of the cells examined. In one particular subculture of MAE28 however, multiple chromosomal rearrangements occurred and the mouse DNA manifested itself in a variety of patterns. Figure 1A depicts a spread with the expected single Xl2 fusion chromosome. In addition, interspecies chromosomal rearrangements such as translocations (Fig. 1B) and insertions (Fig. 1C) were readily detected. The metaphase cell in Fig. 1D contains no mouse chromosomes but does contain mouse DNA in the form of minutes. Finally, growth in selective HAT medium sometimes results in the amplification of the Xl2 mouse fusion chromosome as seen in Fig. 1E. This subculture of MAE28 must have been subjected to stressful or nonoptimal culture conditions, thereby destabilizing the mouse chromosome component. As an alternative approach, as well as a way of generating mouse chromosome-specific probes, we also performed the reciprocal experiment whereby total DNA isolated from several hybrid lines was used on normal female mouse metaphase spreads. Biotinylated DNA from the cell line MalOb (Terao et al., 1988), also containing mouse chromosomes X and 12, clearly decorates the two mouse chromosome pairs in both metaphase (Fig. 1F) and interphase. The signal is not distributed uniformly over the chromosome length, reflecting either an underrepresentation of some sequences or the representation of certain repetitive DNA in the hybrid DNA. Similar chromosome-specific labeling of chromosomes X and 12 was achieved with MAE28 DNA; however, in this case, the centromeres of the other mouse chromosomes were also stained. This may be due to nonoptimal suppression conditions. DNA from cell line MAE19 (Pravtcheva et al., 1981),
129
which contains chromosomes X and 1, also specifically labeled the two corresponding murine chromosome pairs (not shown). The use of high probe concentrations is necessary because of the low percentage of mouse sequences relative to the complete hamster complement in the hybrid cell. The amount of probe DNA required is also influenced by the percentage of cells that contain mouse DNA, and optimal concentrations must be determined empirically for each cell line. At these higher probe concentrations, it becomes necessary to suppress both the hamster and the mouse highly repetitive sequences. For some cell line subcultures that presumably have a very low percentage of cells with mouse chromosomes, chromosome-specific signals are absent but centromere and telomere labeling of chromosomes is observed (data not shown). The telomere labeling is due to unsuppressed hamster telomere repeat sequences, as such results are also obtained when one hybridizes similar concentrations of biotinylated hamster DNA to mouse spreads (Fig. 1G). Furthermore, the signal pattern shown in Fig. 1G is similar to that published for the conserved telomeric repeat sequence (TTAGGG), (Moyzis et al., 1988). This method of chromosome analysis is rapid and the results are easily scored by workers not trained in the subtleties of chromosomal banding. The procedure offers the additional advantage that the mouse component of the cells is identified regardless of the quality of the hybrid line metaphase spreads. Accurate analysis of banding patterns, especially if only fragments of chromosomes are present, relies heavily on well-spread, extended chromosomes of good morphology. A number of different highly repetitive mouse sequences have been hybridized to mouse metaphase spreads to produce distinct banding patterns (A. Boyle and D. Ward, unpublished results). These probes can be used to obtain a banding pattern on only those parts of the chromosomes that are of murine origin. One repeat, which produces a Giemsa-like banding pattern, was hybridized to MAE28 metaphase spreads. (Characterization of this probe will be published elsewhere.) The single mouse chromosome displays a banding pattern identical to that published for the Xl2 fusion chromosome of MAE28 (Pravtcheva et al., 1981) (see Fig.
FIG. 1. (A-E) Biotinylated mouse DNA from cell line Cl27 (8) (5 pg/ml) was hybridized without preannealing with competitor DNA (7) to MAE28 metaphase spreads prepared by standard methods (17). Murine DNA was detected using fluorescein-labeled avidin (green) and the chromosomes were counterstained with propidium iodide (red). (A) The X’r fusion chromosome. (B and C) Interspecies chromosomes exhibiting translocations and insertions, respectively. (D) Minutes containing mouse DNA. The insert shows the same spread with propidium iodide alone, indicating that the signal is DNA and not nonspecific background fluorescence. (E) Amplification of the mouse chromosome. (F-H) Female mouse metaphase spreads from spleen cells were prepared according to Sawyer et al. (14) using 6 ag/ml of concanavalin A and 150 pg/ml of 2-mercaptoethanol for cell stimulation. After 48 h, ethidium bromide (final concentration of 25 &4) and 0.1 &ml of colcemid were added for 30 min. (F) Suppression hybridization was performed using 200 pg/ml of biotinylated MalOb DNA as probe, and both 250 pg/ml of E36 hamster cell line DNA (13) and 200 pg/ml of Cl27 mouse DNA as competitor DNA. Experimental details are described by Lichter et al. (7). (G) Biotinylated E36 hamster DNA (1000 pg/ml) (suppressed with 400 pg/ml of Cl27 mouse DNA and 600 pg/ml of E36 hamster DNA) hybridized to a mouse metaphase spread. (H) Biotinylated mouse repetitive probe (8 pg/ml) hybridized to MAE28 metaphase spread Arrowhead and arrow indicate portions of the fusion chromosome that are derived from chromosomes X and 12, respectively.
130
SHORT
COMMUNICATION
1H). Such hybridization banding methods should prove useful if chromosome identification is required. The use of biotinylated hybrid DNA to decorate specific mouse chromosomes provides an alternative approach to hybrid line analysis. In this case, however, various insertions and rearrangements are not observed and an assessment is based on the extent of labeling of the targeted chromosomes. Nevertheless, appropriate cell lines do provide a valuable source of mouse chromosome-specific probes, as mouse flow-sorted chromosome libraries, unlike human chromosomespecific libraries, are not yet available. Thus, under appropriate suppression conditions, hybrid DNA can be used both to label entire metaphase chromosomes and to delineate interphase chromosome domains, which should also prove useful for topography studies.
interspecific mouse/hamster 105: 169-117.
7.
a.
1.
BOBROW, M., AND CROSS, J. (1974). Differential human and mouse chromosomes in interspecific Nature (London) 251: 77-79.
staining of cell hybrids.
10.
11.
12.
14.
2. CREMER,
T., LICHTER, P., BORDEN, J., WARD, D. C., AND MANUELIDIS, L. (1988). Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum. Genet. 80: 235-
246. 3. DURNAM,
D. M., GELINAS, R. E., AND MYERSON, D. (1985). Detection of species specific chromosomes in somatic cell hybrids. Somatic Cell Mol. Genet. 11: 571-577.
4. HILWIG,
I., AND GROPP, A. (1972). Staining erochromatin in mammalian chromosomes chrome. Exp. Cell Res. 76: 122-126.
5. KOZAK, sequential
of constitutive hetwith a new fluoro-
C. A., LAWRENCE, J. B., AND RUDDLE, F. H. (1977). staining technique for the chromosomal analysis
A of
Exp. Cell Res.
J. E., JANSEN IN DE WAL, N., DIRKS, R. W., BAAS, F., AND VAN DER PLOEG, M. (1987). Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum. Genet. 77: 366-370. LICHTER, P., CREMER, T., BORDEN, J., MANUELIDIS, L., AND WARD, D. C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-234. Lowy, D. R., RANDS, E., AND SCOLNICK, E. M. (1979). Helperindependent transformation by unintegrated Harvey sarcoma virus DNA. J. Viral. 26: 291-299.
9. MANUELIDIS,
13.
REFERENCES
cell hybrids.
6. LANDEGENT,
ACKNOWLEDGMENTS We thank Dr. Dimitrina Pravtcheva and Dr. Frank Ruddle for generously providing us with DNA from the cell lines MalOb and MAE19 and for the E36 cell line. Dr. Arthur Horwich generously supplied the MAE28 cell cultures. P.L. was supported by a stipend from the Deutsche Forschungsgemeinschaft. The research was funded by Grants GM-40115 and GM-41596 from the National Institutes of Health.
somatic
L. (1985). Individual interphase chromosome domains revealed by in situ hybridization. Hum. Genet. 71: 288293. MOYZIS, R. K., BUCKINGHAM, J. M., CRAM, L. S., DANI, M., DEAVEN, L. L., JONES, M. D., MEYNE, J., RATLIFF, R. L., AND WU, J. (1988). A highly conserved repetitive DNA sequence, (TTAGGG),, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. USA 86: 6622-6626. PINKEL, D., STRAUME, T., AND GRAY, J. W. (1986). Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83: 2934-2936. PINKEL, D., LANDEGENT, J., COLLINS, C., FLJSCOE, J., SEGRAVES, R., LUCAS, J., AND GRAY, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 86: 9138-9142. PRAVTCHEVA, D. D., DELEO, A. B., RUDDLE, F. H., AND OLD, L. J. (1981). Chromosome assignment of the tumor-specific antigen of a 3-methylcholanthrene-induced mouse sarcoma. J. Exp. Med. 154: 964-977. SAWYER, J. R., MOORE, M. M., AND HOZJER, J. C. (1987). High resolution G-banded chromosomes of the mouse. Chromosomu
95:350-358. 15.
16.
17.
SCHARDIN, M., CREMER, T., HAGER, H. D., AND LANG, M. (1985). Specific staining of human chromosomes in Chinese hamster X man hybrid cell lines demonstrates interphase chromosome territories. Hum. Genet. 71: 281-287. TERAO, M., PRAVTCHEVA, D., RUDDLE, F., AND MINTZ, B. (1988). Mapping of gene encoding mouse placental alkaline phosphatase to chromosome 4. Somatic Cell Mol. Genet. 14: 211-215. WAHL, G. M., Vrrro, L., PADGETT, R. A., AND STARK, G. R. (1982). Single copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. Mol. Cell. Biol. 2: 308-319.