volume 8 Number is 1980
Nucleic Acids Research
Proteins of metaphase chromosomes and interphase chromatin
Virginia P.Wray', Sarah C.R.Elgin2 and Wayne Wray3
'Dep. Cell Biology, Univ. Texas System Cancer Cent., M.D. Anderson Hospital and Tumor
Inst., Houston, TX 77030, 2 Dep. Biochemistry and Molecular Biology, Harvard Univ.,
Cambridge, MA 02138, and 3 Dep. Cell Biology, Baylor Coll. Medicine, Houston, TX 77030, USA
Received 17 June 1980
ABSTRACT
Metaphase chromosomal and interphase chromatin proteins frcm cells
of two species have been compared by polyacrylamide gel electrophoresis.
Consistent, cannon changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of
ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By
two dimensional gel analysis, the most abundant proteins from chromosomes
of both cell types are similar, suggesting a structural role for these
nonhistone proteins (1).
INTKODUCTICN
Metaphase chromosomes and interphase chromatin represent two different
states of cellular genetic material. Chromosomal proteins which can be
identified as specific to these different structural states of the genomic
ENA may be of particular interest for subsequent studies. Metaphase chromosomes and interphase nuclei of two cell lines were prepared under identical isolation conditions (buffer, ion concentration, etc.) (2). Chroroatin
was then prepared from the nuclei by conventional methods (3). Comparison
of the histories and major nonhistone proteins of metaphase chromosomes and
interphase chromatin from two different cell lines allows a two species
evaluation of parallel content of proteins. The proteins were analyzed by
three comparative gel electrophoresis methods (3-5).
MATERIALS PND METHODS
Chinese hamster ovary (CHD) and human cervical carcinoma ..HeLa) cell
lines were cultured in McCoy's medium 5A with 10% fetal calf serum in a 10%
002 atmosphere. Mitotic cells were detached by shaking monolayers pretreated with 0.06 ug/ml Colcemid (6). Interphase cells were dislodged with a
© IRL Press Umited. 1 Falconberg Court London W1V5FG. U.K.
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rubber policeman.
Metaphase chromosomes and nuclei were prepared by par-
allel isolation procedures in 1.0 M hexylene glycol (2-methyl-2,4-pentanediol, Eastman Organic), 0.5 mM CaCl2, and 0.1 mM PIPES buffer [(piperazineN,N'-ethane sulfonic acid) monosodium monohydrate, Calbiochem] at pH 6.8
(2,7,8).
Cells were broken by nitrogen cavitation at 250 psi. Nuclei were
purified by sedimentation through a 2.1 M sucrose-buffer solution. Chromosomes were purified by centrifugation through a 10-40% sucrose gradient in
a Sorvall SZ-14 zonal rotor (9). The metaphase chromosomes were examined
by using electron
Anderson (10).
microscopy
according
to
Chroniatin was prepared from the purified nuclei as prev-
iously described (11).
follows:
after critical point drying
Protein fractions were prepared and analyzed as
the histories were extracted from the chranatln or suspension of
metaphase chromosomes with 0.2 M H2SO4.
pended in 1% sodium dodecyl sulfate
The remaining pellet was resus-
(SDS), 0.05 M Iris, pH 8, the DNA
removed by centrifugation, and the solution of nonhistone proteins dialyzed
against the sample buffer for gel analysis (12).
Histone extracts were
dialyzed, lyophilized and analyzed on 15% polyacrylamide gels, pH 4.3, in
the presence of urea
(3).
analyzed on polyacrylamide
Nonhistone chromosomal proteins
cylindrical and slab gels
(NHCP) were
(ID-PAGE) or two-
dimensional polyacrylamide gels (2D-PAGE) (3-5).
RESULTS AND DISCUSSION
The NHCP complements of chromosomes and chromatin from CHO and HeLa
cells are presented in the ID-PAGE and absorbance scans shown in Figures
l,a and l,b.
The prominent feature of the comparison is the overall homo-
logy between proteins of the metaphase chromosomes and of the interphase
chromatin.
However, there are quantitative compositional differences be-
tween the interphase chromatin NHCP and metaphase chromosomal NHCP.
sistent differences are observed in both cell lines.
Con-
In the interphase
chromatin, there is an increased amount of two proteins of ca. 65,000 and
68,000 molecular weight ( m ) ( T ). The metaphase chromosomes, on the other
hand, have distinct enrichment of protein bands of ca.
50,000, 90,000-
100,000 and 200,000 MW ( 1 ). Comparison of Figure l,a with Figure l,b indicates greater differences between the NHCP of the two cell types than
between the NHCP of the two interphase and metaphase states. The metaphase
chromosomes of each species have unique NHCP.
Those of HeLa have been pre-
liminarily discussed (13). CHO metaphase chromosomes have a unique protein
of ca.
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58,000 MW (p-58), while those from HeLa have a doublet of proteins
Nucleic Acids Research
200,000 100,000
i
i
50,000
23,000
i
i—"
n—•
200,000 100,000
50,000
25,000
l
1b
I CHO NHCP-M«1aphas«
II CHO NHCP-Int«rpho»»
"•. .-S\
•"•-•-
J
n -•
Fig. 1: Nonhistone chraraosanal proteins of isolated metaphase chromosomes
and interphase chronatin. Proteins in samples were resolved according to
molecular weight in 7.5%, 0.6 x 10 an polyacrylamide gels (4). Gel origin
is to the left. Protein bands of interest are indicated by ( i ) and ( » ).
a. HeLa cells, b. O C cells.
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of ca. 35,000-37,000 HW and a single polypeptide of ca. 29,000 HW. Essentially no difference is noted in the histone patterns of CH0 and HeLa raetaphase chromosomes or interphase chromatin (Fig. 2 ) . A small amount of H3
dimer is observed; however, there was no indication that either disulfide
linked H3 multimers or H3-NHCP complexes were preferentially formed in the
metaphase state, as has been previously suggested (14). Similar results
on H3 have been reported by other research groups (15).
Analysis of the metaphase NHCP by 2D-PASE further demonstrated both
inter species horology and species variation in predominant proteins
of OH and HeLa cell chromosones. The gel shown in Figure 3 is a 2-D
PAGE separation of CHO metaphase chromosomal NHCP. Samples of Hela metaphase chromosomal NHCP and bovine brain tubulin were run on duplicate gels
under the same conditions. The position of the major HeLa NHCP spots are
indicated by circles drawn on the figure. Some of the CHO and HeLa NHCP
have identical molecular weights and isoelectric points while others are
H3d
I
n
m
12
HI
H«LO
HtLa
CHO
CHO
H2-3
HntOTM
HoKXM! HUtarm Hbtoau -
Fig. 2: Histones of metaphase chromosones and interphase chromatin of HeLa
and CUD cells. Samples were resolved into component histones by standard
methods (3). Gels run left to right.
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«
IFSDS
oo
56-
i
4542-
1
oo> oo •
1*
25- —»
Fig. 3: Two-dimensional separation of CE© metaphase chromosomal proteins.
Sample protein was separated by electrophoresis on a 1.5 mn x 8.5 cm, 9.5%
polyacrylamide gel according to the O'Farrell method (5). Protein standards
of bovine serum albumin, 68,000 MW; bovine brain tubulin, 56,000 MW; rabbit
muscle actin, 45,000 MW; liver alcohol dehydrogenase, 42,000 MW; and alphachymotrypsinogen, 25,700 MW were applied in a separate well adjacent to the
first dimension cylindrical gel and run in the second dimension. The positions of migration of the tubulin monomers are indicated by hash lines.
Migration of Hel^i metaphase chromosome NHCP are indicated by circles drawn
on the figure. Proteins unique to Hela fractions are indicated by arrows.
unique to the cell type.
are marked by arrows.
Proteins seemingly unique to HeLa cell chromosomes
Positions of migration of the tubulin monomers are
indicated by the spots containing slashed lines.
The amount of protein
applied to the gels was selected to demonstrate the detection of only the
most abundant proteins of the total population within the respective samples.
The majority of the proteins are acidic with an apparent pi of less
than 6.5.
The prominent 58,000 and 50,000 MW proteins of the CHO chromosome
fraction have nobilities different from those of tubulin and actin as shown
by both 1D-PM2: slab gel analysis and 2D-PAGE analysis.
Figure 4 shows a
1D-PAGE comparison of total CHO chromosomal proteins to molecular weight
standards containing preparations of purified
bovine
brain tubulin and
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-94
-68
-29
Fig. 4; Comparison of CHO metaphase chranosanal proteins to standard tubulin and actin markers. Metaphase chronosornes were prepared as described
and resolved by electrophoresis on 0.75 x 9 x 8.5 an slabs of
9.5% polyacrylamide (4). Numbers represent Molecular Weight x 10~ 3 . Standard
protein markers are phosphorylase a, 94,000 MW: bovine senm albunin,
68,000 MW; bovine brain tubulin, 56,000 MW; fumarase, 49,000 MW; rabbit
muscle actin, 45,000 MW; liver alcohol dehydrogenase, 42,000 MW; and carbonic anhydrase, 29,000 MW.
rabbit muscle actin. Neither chromosomal protein p-58 nor protein p-50
comigrate with the protein standards. The p-58 protein has an apparent
isoelectric point different from either of the tubulin isomers (see Figure
3) which further substantiates that p-58 is not tubulin. These findings
are in contrast to the previous suggestion that tubulin and actin may be
the two major protein constituents of CHO chromosomes (16). The biochemical characteristics of band 58 will be reported elsewhere.
It has been shown previously that metaphase chromosomes isolated by
the procedure used here exhibit approximately the same mass ratios of
DNA:histones: NHCP:RNA as does isolated interpnase chromatin (1:0.9-1.2:
0.3-1.2:0.1) (2). Consequently, relatively small differences in the protein populations of the two different cell cycle stages might be anticipated. The similarity of the NHCP patterns of interphase chromatin and metaphase chromosomes supports the concept that the NHCP are a defined class
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of proteins in tight association with the ENA throughout the cell cycle.
However, studies by others have indicated that 3-6% of the chromosomal
proteins of interphase chroraatin could be cytoplasmic material adventitiously adsorbed during the isolation procedure
(17,18).
The following
experiment demonstrates that metaphase chromosomes isolated by the WrayStubblefield procedure may be easily freed from adventitiously adsorbed
cytoplasmic proteins after isolation.
cells was prepared
A nuclei-free cytosol from lysed
from a randomly growing CHO cell culture prelabeled
with [14C]leucine for one generation.
somes were then incubated
Isolated nonradioactive CHO chrano-
in the radioactive cytosol for 30 min.
chromosomes were purified through buffered sucrose gradients.
The
Autoradic—
graphic analysis demonstrated there were no exposed silver grains associated with the chromosomes after six months exposure.
We estimate that
this type of experiment will detect less than 1% contamination from adventitiously adsorbed proteins.
The interphase chromatin was prepared from unsynchronized cells, and
consequently represents an average of all of the cell states including
about 3.0% in metaphase.
The contribution of any special "mitotic proteins"
to the total interphase protein complement would be undetectable by protein
detection methods used for this study.
Parallel changes in the NHCP populations of chromatin compared to raetaphase chranosomes of the two cell types would suggest presence of proteins
specific to the raetaphase or interphase state.
The 50,000 MW protein ob-
served in this study and the scaffold protein of Adolph, et al. are possibly
the same since they have similar molecular weights
(19). However, the
scaffold material has two polypeptides of equal abundance whereas these
studies indicate only a single predominent protein.
The 200,000 MW NHCP
has a mobility equal to that of muscle myosin, but has yet to be shown to
be rayosin.
The NHCP pattens of interphase chromatin andraetaphasechromosomes are
basically similar for a given cell type.
These studies demonstrate, how-
ever, that there are several quantitative differences between the NHCP
patterns of
chromosomes and chromatin.
These quantitative
differences
were observed in both CHO and HeLa cells which suggests that the proteins
showing altered content in the metaphase chromosomes nay have structural
roles in the organization of the metaphase chromosome.
There are also differences in protein content which appear to be cell
type specific.
These differences can be observed in both single and two
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dimensional gels. The most apparent difference between HeLa and CHO metaphase chromosomal proteins i s the 35,000-37,000 MW components present in
HeLa chranoecines. By nultisanple ID-slab gel analysis, the relative migration of these HeLa proteins i s intermediate to that of major CHO proteins
of the same general gel region. The 29,000 MW HeLa protein migrates in
advance of comparable CHO proteins. Although there i s a protein of approximate 58,000 MW present in HeLa cell chromosomes, this protein does not
nave an apparent pi corresponding to the 58,000 MW protein of the CHO chromosomes. Nuclei and chroraosme fractions from the two cell types were prepared and analyzed simultaneously to minimize sources of variance. Puther
studies will include more sensitive analytical methods and chromosomes of
other cell types to expand these results. The role of these proteins in
interphase and metaphase states i s also under investigation.
ACKNOWLEDGEMENTS
This work was supported by grants No. 305 from t h e Jane coffin Childs
Memorial Fund for Medical Research and GM-20779 from t h e National I n s t i t u t e s
of Health ( t o S.C.R.E.), Cft-18455, GM-26415 and NSF PCM7-05428 (toW.W.) and
CA-23527 and BR-5511-17 ( t o V.P.W). W.W. i s the r e c i p i e n t of Research Cancer
Development Award CA-00532 from the National Cancer I n s t i t u t e .
Address communication to: Dr. Wayne Wray, Department of Cell Biology, Baylor College of
Medicine, 1200 Moursund, Houston, TX 77030, USA
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