We have analyzed the organization of the homogeneously staining regions (HSRs) in chromosomes from a methotrexate-resistant mouse melanoma cell line. Fluorescence in situ hybridization techniques were used to localize satellite DNA sequences and the amplified copies of the dihydrofolate reductase (DHFR) gene that confer drug-resistance, in combination with immunofluorescence using antibody probes to differentiate chromatin structure. We show that the major DNA species contained in the HSRs is mouse major satellite, confirming previous reports, and that this is interspersed with DHFR DNA in an alternating tandem array that can be resolved at the cytological level. Mouse minor satellite DNA, which is normally located at centromeres, is also distributed along the HSRs, but does not appear to interfere with centromere function. The blocks of major satellite DNA are coincident with chromatin domains that are labelled by an autoantibody that recognizes a mammalian homologue of Drosophila heterochromatin-associated protein 1, shown previously to be confined to centric heterochromatin in mouse. An antiserum that specifically recognizes acetylated histone H4, a marker for active chromatin, fails to bind to the satellite DNA domains, but labels the intervening segments containing DHFR DNA. We can find no evidence for the spreading of the inactive chromatin domains into adjacent active chromatin, even after extended passaging of cells in the absence of methotrexate selection.
REFERENCES
Alt
F. W.
, Kellems
R. E.
, Bertino
J. R.
, Schimke
R. T.
(1978
). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells.
J. Biol. Chem
253
, 1357
–1370
.Bertino
J. R.
, Donohue
D. M.
, Simmons
B.
, Gabrio
B. W.
, Silber
R.
, Huennekens
F. M.
(1963
). The induction of dihydrofolate reductase activity in leukocytes and erythrocytes in patients treated with amethopterin.
J. Clin. Invest
42
, 466
–475
.Biedler
J. L.
, Spengler
B. A.
(1976
). Metaphase chromosome anomaly: association with drug resistance and cell-specific products.
Science
191
, 185
–187
.Biedler
J. L.
, Melera
P. W.
, Spengler
B. A.
(1980
). Specifically altered metaphase chromosomes in anti-folate-resistant Chinese hamster cells that overproduce dihydrofolate reductase.
Cancer Genet. Cytogenet
2
, 47
–60
.Bostock
C. J.
, Clark
E. M.
, Harding
N. G. L.
, Mounts
P. M.
, Tyler-Smith
C.
, van Heyningen
V.
, Walker
P. M. B.
(1979
). The development of resistance to methotrexate in a mouse melanoma cell line. I. Characterization of the dihydrofolate reductases and chromosomes in sensitive and resistant cells.
Chromosoma
74
, 153
–177
.Bostock
C. J.
, Clark
E. M.
(1980
). Satellite DNA in large marker chromosomes of methotrexate-resistant mouse cells.
Cell
19
, 709
–715
.Brinton
B. T.
, Heintz
N. H.
(1995
). Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification.
Chromosoma
104
, 143
–151
.Butner
K.
, Lo
C. W.
(1986
). Modulation of tk expression in mouse pericentromeric heterochromatin.
Mol. Cell. Biol
6
, 4440
–4449
.Carman
M. D.
, Schornagel
J. H.
, Rivest
R. S.
, Srimatkandada
S.
, Portlock
C. S.
, Duffy
T.
, Bertino
J. R.
(1984
). Resistance to methotrexate due to gene amplification in a patient with acute leukaemia.
J. Clin. Oncol
2
, 16
–20
.Chang
S. E.
, Littlefield
J. W.
(1976
). Elevated dihydrofolate reductase messenger RNA levels in methotrexate-resistant BHK cells.
Cell
7
, 391
–396
.Crouse
G. F.
, Simonsen
C. C.
, McEwan
R. N.
, Schimke
R. T.
(1982
). Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells.
J. Biol. Chem
257
, 7887
–7897
.Csordas
A.
(1990
). On the biological role of histone acetylation.
Biochem. J
265
, 23
–38
.Dolnick
B. J.
, Berenson
R. J.
, Bertino
J. R.
, Kaufman
R. J.
, Nunberg
J. H.
, Schimke
R. T.
(1979
). Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells.
J. Cell Biol
83
, 394
–402
.Eissenberg
J. C.
, James
T. C.
, Foster-Hartnett
D. M.
, Hartnett
T.
, Ngan
V.
, Elgin
S. C. R.
(1990
). Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster.
Proc. Nat. Acad. Sci. USA
87
, 9923
–9927
.Fantes
J. A.
, Bickmore
W. A.
, Fletcher
J. M.
, Ballesta
F.
, Hanson
I. M.
, van Heyningen
V.
(1992
). Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization.
Am. J. Hum. Genet
51
, 1286
–1294
.Foreman
P. K.
, Hamlin
J. L.
(1989
). Identification and characterization of a gene that is co-amplified with dihydrofolate reductase in a methotrexate resistant CHO cell line.
Mol. Cell. Biol
9
, 1137
–1147
.Gooderham
K.
, Jeppesen
P.
(1983
). Chinese hamster metaphase chromosomes isolated under physiological conditions: a partial characterization of associated non-histone proteins and protein cores.
Exp. Cell Res
144
, 1
–14
.Hebbes
T. R.
, Thorne
A. W.
, Crane-Robinson
C.
(1988
). A direct link between core histone acetylation and transcriptionally active chromatin.
EMBO J
7
, 1395
–1402
.Hebbes
T. R.
, Thorne
A. W.
, Clayton
A. L.
, Crane-Robinson
C. R.
(1992
). Histone acetylation and globin gene switching.
Nucl. Acids Res
20
, 1017
–1022
.Heintz
N. H.
, Hamlin
J. L.
(1982
). An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments.
Proc. Nat. Acad. Sci. USA
79
, 4083
–4087
.Henikoff
S.
(1990
). Position-effect variegation after 60 years.
Trends Genet
6
, 422
–426
.Horns
R. C.
, Dower
W. J.
, Schimke
R. T.
(1984
). Gene amplification in a leukaemic patient treated with methotrexate.
J. Clin. Oncol
2
, 2
–7
.Horz
W.
, Altenburger
W.
(1981
). Nucleotide sequence of mouse satellite DNA.
Nucl. Acids Res
9
, 683
–696
.James
T. C.
, Elgin
S. C. R.
(1986
). Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.
Mol. Cell. Biol
6
, 3862
–3872
.Jeppesen
P.
, Mitchell
A.
, Turner
B.
, Perry
P.
(1992
). Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes.
Chromosoma
101
, 322
–332
.Jeppesen
P.
, Turner
B. M.
(1993
). The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression.
Cell
74
, 281
–289
.Jones
K.
(1970
). Chromosomal and nuclear location of mouse satellite DNA in individual cells.
Nature
225
, 912
–915
.Kellems
R. E.
, Alt
F. W.
, Schimke
R. T.
(1976
). Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma S-180 cells. In vitro translation and characterization of folate reductase mRNA.
J. Biol. Chem
251
, 6987
–6993
.Koch
J. E.
, Kølvraa
S.
, Petersen
K. B.
, Gregersen
N.
, Bolund
L.
(1989
). Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ.
Chromosoma
98
, 259
–265
.Langer-Safer
P. R.
, Levine
M.
, Ward
D. C.
(1982
). Immunological method for mapping genes on Drosophila polytene chromosomes.
Proc. Nat. Acad. Sci. USA
79
, 4381
–4385
.Masumoto
H.
, Masukata
H.
, Muro
Y.
, Nozaki
N.
, Okazaki
T.
(1989
). A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite.
J. Cell Biol
109
, 1963
–1973
.Mitchell
A.
, Jeppesen
P.
, Hanratty
D.
, Gosden
J.
(1992
). The organization of repetitive DNA sequences on human chromosomes with respect to the kinetochore analysed using a combination of oligonucleotide primers and CREST anticentromere serum.
Chromosoma
101
, 333
–341
.Mitchell
A. R.
, Nicol
L.
, Malloy
P.
, Kipling
D.
(1993
). Novel structural organization of a Mus musculus DBA/2 chromosome shows a fixed position for the centromere.
J. Cell Sci
106
, 79
–85
.Mitchell
P. J.
, Carothers
A. M.
, Han
J. H.
, Harding
J. D.
, Kas
E.
, Veniola
L.
, Chasin
L. A.
(1986
). Multiple transcription start sites, DNase I hypersensitive sites, and an opposite strand exon in the 5region of the CHO DHFR gene.
Mol. Cell. Biol
6
, 425
–440
.Nicol
L.
, Jeppesen
P.
(1994
). Human autoimmune sera recognize a conserved 26 kD protein associated with mammalian heterochromatin that ishomologous to heterochromatin protein 1 of Drosophila.
Chrom. Res
2
, 245
–253
.Nunberg
J. H.
, Kaufman
R. J.
, Schimke
R. T.
, Urlaub
G.
, Chasin
L. A.
(1978
). Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line.
Proc. Nat. Acad. Sci. USA
75
, 5553
–5556
.Pardue
M. L.
, Gall
J. G.
(1969
). Molecular hybridization of radioactive DNA to the DNA of cytological preparations.
Proc. Nat. Acad. Sci. USA
64
, 600
–604
.Pietras
D. F.
, Bennet
K. L.
, Siracusa
L. D.
, Woodworth-Gutai
M.
, Chapman
V. M.
, Gross
K. W.
, Kane-Haas
C.
, Hastie
N. D.
(1983
). Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.
Nucl. Acids Res
11
, 6965
–6983
.Pluta
A. F.
, Saitoh
N.
, Goldberg
I.
, Earnshaw
W. C.
(1992
). Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere.
J. Cell Biol
106
, 1081
–1093
.Schimke
R. T.
, Kaufman
R. J.
, Nunberg
J. H.
, Dana
S. L.
(1979
). Studies on the amplification of dihydrofolate reductase genes in methotrexate-resistant cultured mouse cells.
Cold Spring Harbor Symp. Quant. Biol
43
, 1297
–1303
.Singh
P. B.
, Miller
J. R.
, Pearce
J.
, Kothary
R.
, Burton
R. D.
, Paro
R.
, James
T. C.
, Gaunt
S. J.
(1991
). A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants.
Nucl. Acids Res
19
, 789
–794
.Talarico
D.
, Peverali
A. F.
, Ginelli
E.
, Meneveri
R.
, Mondello
C.
, Valle
G. D.
(1988
). Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.
Mol. Cell. Biol
8
, 1336
–1344
.Trent
J. M.
, Buick
R. N.
, Olsen
S.
, Horns
R. C.
, Schimke
R. T.
(1984
). Cytologic evidence for gene amplification in methotrexate resistant cells obtained from a patient with ovarian adenocarcinoma.
J. Clin. Oncol
2
, 8
–15
.Turner
B. M.
, Fellows
G.
(1989
). Specific antibodies reveal ordered and cell-cycle-related use of histone-H4 acetylation sites in mammalian cells.
Eur. J. Biochem
179
, 131
–139
.Turner
B. M.
(1991
). Histone acetylation and control of gene expression.
J. Cell Sci
99
, 13
–20
.Wong
A. K. C.
, Rattner
J. B.
(1988
). Sequence organization and cytological localization of the minor satellite of mouse.
Nucl. Acids Res
16
, 11645
–11661
.Wreggett
K. A.
, Hill
F.
, James
P. S.
, Hutchins
A.
, Butcher
G. W.
, Singh
P. B.
(1994
). A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin.
Cytogenet. Cell Genet
66
, 99
–103
.
This content is only available via PDF.
© 1996 by Company of Biologists
1996
-PolarityCFP.png?versionId=4696)
