Download The abundance and cell cycle dependent expression of the mRNA

Volume 15 Number 8 1987
Nucleic Acids Research
Cell cycle regulated synthesis of an abundant transcript for human chromosomal protein
HMG-17
Michael Bustin, Ninnolini Soarcs, David Landsman, Thyagarajan Srikantha and James M.Collins*
Laboratory of Molecular CarcinogeDesis, National Cancer Institute, National Institutes of Healdi,
Bethesda, MD 20892 and *Department of Biochemistry, Medical College of Virginia, Richmond, VA
23298-0001, USA
Received December 24, 1986; Revised and Accepted March 12, 1987
ABSTRACT.
The abundance and cell cycle dependent expression of the mRNA
for human nonhistone protein HMG-17 were studied in synchronized
HeLa cells. Slot blot analysis indicates that the HMG-17 mRNA is
a very abundant message, significantly more so than histone or
actin mRNA. RNA prepared from tissue culture cells contains
higher amounts of HMG-17 transcripts than RNA prepared from liver
suggesting a correlation between the rate of cell division and
HMG-17 mRNA levels. HMG-17 mRNA is present in the cells throughout the cell cycle however there is a significant increase in the
mRNA levels late in S phase suggesting that the protein is
deposited on chromatin after nucleosome assembly. Synthesis of
the HMG-17 transcript is not coupled to DNA replication suggesting that the cell cycle related expression during late S phase is
regulated in a different manner from that of the nucleosomal
histones.
INTRODUCTION.
Chromatin regions containing transcribable genes have an
altered chromatin conformation which is more susceptible to
digestion with DNasel than the bulk of the genome. It has been
suggested that nonhistone chromosomal proteins HMG-14 and HMG-17
may be involved in maintaining transcribable genes in this
altered chromatin conformation (1,2) however, this suggestion is
still controversial (reviewed in 3 ) . Nonhistone chromosomal
proteins HMG-14 and HMG-17 are present in the nuclei of most
higher eukaryotic cells. In the nucleus, the primary binding site
of these proteins is the core particle itself (4). The amount of
HMG-14 and HMG-17 present in a cell is sufficient to bind only
about 10% of the nucleosomes yet reconstitution experiments
demonstrate that each nucleosome has two binding sites for the
proteins (5,6,7). Support for the putative regulatory role of
these proteins comes from experiments which indicated that: 1.
the preferential DNasel sensitivity of active chromatin is
dependent on the presence of HMG-14 and HMG-17 (2), 2.
microinjection of antibodies to HMG-17 into the cell nucleus
inhibits transcription (8), 3. antibodies to HMG-14 preferential3549
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ly bind to transcriptionally active regions of polytene chromosomes (9), 4. the proteins bind preferentially to salt-stripped
nucleosomes containing transcriptionally active genes (6) and 5.
antibodies to HMG-17 bind preferentially to chromatin fragments
enriched in transcriptionally active genes (10,11).
Studies on the chemical properties and chromosomal location of
HMG-17 have, so far, failed to clarify its cellular role. The
availability of molecular probes for HMG-17 (12) opens new
approaches for such studies. It is of particular interest to
compare the regulation of the gene expression of HMG-17, a
nonhistone protein, to that of the histones, which is cell cycle
related (13). Recently we reported that the HMG-17 transcript,
which in humans is encoded by at least one member of a multigene
family, has a very long AT-rich untranslated 3' end and an
exceedingly GC-rich untranslated 5 1 region (12). In the present
manuscript we investigate the abundance of the HMG-17 message in
human cells and study its expression during the cell cycle. We
find that the HMG-17 transcript is very abundant and that its
expression is cell cycle regulated.
MATERIALS AND METHODS.
Cell culture and synchronization.
HeLa cells at a concentration of 0.5xl06 cells/ml were
maintained in spinner culture at 37°. Cells were fed every 48 hr.
with Joklik's modified Eagle's minimal essential medium containing 10% fetal calf serum and 1.25 ug/ml of Fungizone (14). For
synchronization, cells were maintained on media containing 2mM
thymidine for 14hr, centrifuged, resuspended in fresh medium
without thymidine, and allowed to grow for 9 hr. The cells were
exposed to a second 2mM thymidine block for 14 hr, then released
from the block by resuspension in fresh medium. Under these
conditions greater than 96* of the cells were initially at the
Gl/S boundary, as judged by the subsequent movement of the cells
through the S, G2, M, and Gl phases of the cell cycle. The
movement of the cells through the cycle was monitored by flow
cytometry of cellular DNArpropidium iodide fluorescence and the
percentages of the cells in each phase of the cycle were determined by computer analysis of the DNA distribution as previously
described (14).
RNA preparation.
Total cellular RNA was extracted from the cell lines and from
tissues by the guanidinivun thiocyanate method (15). Poly
A+-enriched RNA was obtained by chromatography on oligo-dT
columns (16).
Northern and slot blot analysis.
For slot blot analysis the RNA was denatured in 3 3%
formaldehyde, 7xSSC at 65° for lOmin and applied to Zetabind
filters (AMF, Meriden, Conn). For Northern analysis the RNA
samples were treated with formaldehyde-formamide solutions and
electrophoresed in denaturing formamide gels (15). The gels were
washed briefly with H20, the RNA nicked by short incubation in
0.2N NaOH and transferred to Zetabind filters by the Southern
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procedure (17). The Zetabind filters were treated as recommended
by Church and Gilbert (18). Hybridizations were done in 1% bovine
serum albumin, 7% sodium dodecyl sulfate, 0.5H sodium phosphate
buffer pH7 and lmM EDTA at 65° for 18hr. The filters were washed
exactly as recommended by Church and Gilbert (18).
DNA probes
Plasmids were isolated from the lysates of chloramphenicol
treated cultures by banding in CsC12/ethidium bromide gradients.
The HMG-17 probe used to detect the mRNA was prepared by excising
the insert from plasmid pH17c with ECoRl. This insert constitues
essentially the full length HMG-17 cDNA (12). The HMG-14 mRNA was
detected with the full length cDNA excised from plasmid pH14c
(19). Histone mRNA was detected with an insert excised from
plasmid plO8A (20), actin mRNA was detected using the insert
excised from plasmid pAcH8H. The DNA fragments were nick translated and used for Northern or slot blot analysis as described in
the previous paragraph. For SI protection assays the insert
obtained from pH17c was digested with Pstl and the 295 bp 5 1
fragment isolated by gel electrophoresis. The fragment was
dephosphorylated and end labelled according to Maniatis (15).
Various RNA samples were hybridized to the end-labelled fragment
treated with SI nuclease and analysed according to Berk and Sharp
(21). The digestion mixtures were analysed on 6% polyacrylalmide,
8M urea gels (15).
Protein labelling in HeLa cells.
Exponentially growing HeLa cells were grown for 15 min in
lysine free media, containing 5% dialysed fetal calf serum, 0.2%
glutamine and O.lmCi per ml of 3H lysine. The cells were washed
in serum free media and nuclei isolated by homogenization in
0.05H Tris pH7.5, 0.25M sucrose, 0.025M KC1, 2mM MgCl, lmM PMSF
(phenylmethylsulfonyl fluoride), 1% trasylol (Sigma) and 0.5%
Triton X-100. HMG proteins were isolated from the nuclei by the
PCA (perchloric acid) procedure, the extract precipitated with 6
volumes of acetone, the precipitate analysed by electrophoresis
on 18% polyacrylamide gels containing sodium dodecyl sulfate and
the position of the radioactive proteins visualized by
fluorography (22).
RESULTS.
HMG-17 mRNA is an abundant message.
The human genome contains a multigene family which codes for a
single-size HMG-17 mRNA. Sequence analysis (12) reveals that the
transcript is unusual in that the open reading frame constitutes
only 25% of the transcript (see fig 1), the 3 1 untranslated
region is extremely long and rich in AT residues, and the 5 1
untranslated region is rich in GC residues. The full length
HMG-17 cDNA and fragments derived from it were used to determine
the relative abundance of this message in HeLa cells. The data
presented in fig 2A indicates that, in HeLa cells, HMG-17 mRNA is
very abundant. In these experiments, samples of poly A+ enriched
RNA, total RNA and standards of known amounts of plasmid DNA were
slot blotted onto a Zetabind filter. The filter was cut into
several sections and each section tested with a nick translated
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I
I
'
h is z I I
^
—
200
I
400
600
I
h
I II
1
H
1 H
800
1000
-\ b*x pairs
Fig. 1. Restriction map of the HMG-17 cDNA used for studies on
the transcriptional regulation of the HMG-17 gene. The open
reading frame of the cDNA is indicated by the shaded region. The
transcript is characterized by long untranslated 3' region and a
GC rich untranslated 5'region. The 295 bp long EcoRl-Pstl fragment was used for the SI nuclease protection experiments described below.
probe specific for a known gene product. The radioactivity bound
was detected by autoradiography (fig 2A), the radioactive slots
cut from the filter and the number of 3 2 P counts bound to each
slot was determined by liquid scintillation. The counts bound to
the known amounts of plasmid DNA were used to construct a standard curve relating the number of counts to the amount of DNA
applied to the filters. These were used to normalize for the
difference in specific activities and hybridization efficiency of
the various probes thereby allowing a meaningful comparison of
the relative amounts of the specific mRNAs in the samples studied. The results presented in Tablel indicate that HMG-17 mRNA is
about 32-fold more abundant than actin mRNA and 15-fold more
abundant than histone H4 or HMG-14 mRNA.
The abundance of the HMG-17 mRNA is not due to non-specific
binding of the probe to other cellular RNAs since, as we previously reported (12), Northern analysis of total cellular RNA
indicated that this probe binds only to a single-size mRNA
species, about 1200 nucleotides long. Furthermore, SI nuclease
protection assays (fig 2B) also indicate that the cDNA indeed
corresponds to a true cellular transcript. The SI protection
assays were done with the EcoRl-Pstl fragment derived from the 5'
region of the transcript (see fig 1 ) . The fragment was
end-labelled with 3 2 P and exposed to SI nuclease either in the
absence of RNA or in the presence of total RNA prepared from
either HeLa cells or rat liver. The results (fig2B) reveal
specific protection of the end-labelled fragment by the human
RNA. The RNA derived from the human cells protected the fragment
about 10 fold better than the RNA derived from rat attesting to
the specificity of the reaction and to the fact that the cDNA
isolated corresponds to a true transcript which is present in
human cells in significant quantities. This point was important
to establish since the protein is encoded by a multigene family
and therefore it is possible that the species isolated and
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1 2 3 4 bp
T.
^P
4 9 Liver
^
^
H
5/ig
HeLa
Fig. 2. Quantitation of HMG-17 mRNA in HeLa cells. A^ Slot blot
analysis. Two different preparations of polyA-enriched RHA and
one of total RNA were slotblotted on a Zetabind membrane, together with appropriate plasmids which served as standards for
quantitative hybridization. The odd numbered lanes (1,3,5,7)
contained the RNA (A, 0.7ug poly A+; B, 0.8ug of another poly A+
preparation; c, 12ug of total RNA). The even numbered lanes
(2,4,6,8) contained plasmids (A, ing; B, 0.5ng; C, 0.25ng). The
filter was cut into 4 pieces and probed with nick translated
probes prepared from the plasmids which were applied to the
filter. The mRNA detected by the probe is indicated on the right.
B. SI protection experiments. The 295bp EcoRl-Pst 1 fragment (see
fig 1.) was end labelled and incubated with 1,4, no RNA; 2, 20ug
human RNA: or 3, 20 ug rat RNA. Samples 1,2,3 were incubated with
SI. Sample 4 contained no SI. Afer SI digestion the samples were
run on a 5' polyacrylamide and autoradiographed (see methods
section).
C. Quantitation of HMG-17 mRNA in various cells. Either 5 or 15
ug total RNA isolated from human liver or HeLa cells, were
slotblotted on Zetabind filters and probed for their content of
HMG-17 mRNA.
seguenced is in fact a minor constituent of total cellular HMG-17
RNA. Since the presence of HMG-17 has been correlated with
transcriptionally active chromatin, we next tested whether
rapidly dividing, transcriptionally active, cells contain more
HMG-17 mRNA than slowly dividing cells. Equal amounts of RNA
prepared from either human liver or HeLa cells were probed with
HMG-17 cDNA. Under conditions of high stringency, the signal
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TABLE I. Quantisation of mRNAs in HeLa cells.
pg mRNA 2 .
PROBE.
CPM
CPM/ng 1
Of PROBE.
Actin
357
Histone 153
pH14c
142
pH17c
1207
Ill
185
236
3550
3200
600
650
340
RATIO.
1
2
2
32
1
The CPM/ng probe was determined from a standard curve. (See fig
2A for a portion of the curve). This value is the relative
specific activity of the probe.
2
Total pg of RNA in a sample was determined by dividing the
number of CPM in the slot by the specific activity of the probe,
i.e. CPM/ng probe.
1
2
3
H1HMG-1,2HMG-14HMG-17-
Fig. 3. HMG-17 turnover in HeLa cells. HeLa cells were pulse labelled with 3H lysine and the nuclear proteins extractable by 5%
PCA examined by electrophoresis in 18% polyacrylamide gels
containing sodium dodecyl sulfate. Lane 1, Coommassie blue stain
indicating the position and the relative quantity of the proteins
present in the 5% PCA extract; lane 2, Tritiated HMG-1, HMG-2 and
HMG-17 radioactive markers, lane 3, fluorogram of the 5% PCA
extract from the labelled cells. The position of histone HI and
of the major HMG proteins is indicated on the left.
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obtained from the human samples was very strong (fig 2C). The
relative content of HMG-17 mRNA in the two human samples was
determined by scanning the autoradiograms and integrating the
area under each peak. The data indicates that HeLa RNA contained
approximately 6 times more HMG-17 mRNA than the RNA extracted
from human livers. Since the levels of HMG-17 RNA are also high
in A549 and MCF-7 cells (12) these results seem to indicate that
tissue culture cells may have a higher content of HMG-17 mRNA
than cells obtained from an organ where most of the cells are in
the Go stage.
HMG synthesis in HeLa cells.
The abundance of the HMG-17 transcript in the tissue culture
cells is surprising since the amount of HMG-17 protein in a cell
is significantly lower than that of actin or any of the histones
(23). The low level of HMG-17 protein in cells could be due to
rapid protein turnover as indicated by the pulse labelling
experiments presented in fig 3. Exponentially growing HeLa cells
were labelled with 3 H Lysine for 15 minutes and the HMG proteins
and histone HI present in the nuclei extracted with 5% PCA. The
extract was electrophoresed on polyacrylamide gels, the protein
content visualized by staining the gels with Coommassie blue and
the relative level of 3H-lysine incorporated determined by
fluorography. A scan of the Coommassie blue pattern presented in
lane 1 of fig 3 indicated that the amount of HMG-17 was less than
5% of the amount of HI in the cells. The ratio of HMG-14 to
HMG-17 was 0.5; the HMG-l+HMG-2 to HMG-17 ratio was 2.5. In the
autoradiographs (lane 3) the HI to HMG-17 ratio was 3, the HMG-14
to HMG-17 ratio was 0.10 and the HMG-1+2 to HMG-17 ratio was 0.4.
The lysine content of all these proteins is similar therefore the
results indicate that HMG-17 incorporates lysine more rapidly
than the other proteins in the extract, suggesting a relatively
high turnover rate for the protein. We wish to emphasize, however, that the data represent a single time point and that further
experiments are necessary for accurate determination of the
relationship between mRNA stability and protein turnover rate.
Transcription during the cell cycle.
The relative levels of transcription of the HMG-17 gene during
the cell cycle in HeLa cells were determined using cells synchronized by the double thymidine block method (14). Following
release from the second thymidine block, a portion of the cells
was taken at various times for determination of the DNA:propidium
fluorescence. The progression of the synchronized HeLa cells,
through the cell cycle was monitored by flow cytometry of the
fluorescent cells (fig.4). The time after release from the double
thymidine block is indicated at the upper left side of each
panel. Computer analysis of the DNA distribution in each sample
indicates that the percentage of cells in each phase of the cell
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(S)
i
i
LLJ
o
o
UJ
CD
DNA CONTENT
Fig 4. Cell cycle progression of synchronized cells. At the times
indicated in the upper left of each panel, portion of HeLa cells,
synchronized by a double thymidine block, were analysed for their
DNA content by flow cytometry , as described in the methods
section. The modal channel numbers for cells with Gl and G2M DNA
contents were 40 and 80 respectively, and are indicated at the
bottom of the figure by 1 and 2 respectively. The number of cells
in the maximum channel was 1000 for each determination. The modal
channel numbers of each peak of the DNA distributions were: 0-hr,
40; 2-hr, 42; 4-hr, 51; 6-hr, 59; 8-hr, 71; 10-hr, 40 and 80;
14-hr, 40 and 80; 16-hr, 40; 22-hr, 40.
cycle were as follows: 0-hr, 98% Gl/S boundary; 2-hr, 88*
early-S; 4-hr 96% mid-S, 6-hr, 97% mid-S; 8-hr, 92% late-S;
10-hr, 78% G2M, 11% S, 11% Gl; 14-hr, 84% Gl, 26% G2H; 16-hr, 93%
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B
-HMG
— Histone H4
0 3 8 14 16 18
Fig. 5. Northern analysis of the cell cycle expression of
HMG-17. A. 12ug of RNA isolated from cells at various times after
release from the double thymidine block (indicated) were fractionated on 1% agarose-fonnaldehyde gels and transferred to
Zetabind filters. B. lOug RNA extracted from exponentially
growing cells and 15 ug of RNA extracted from hydroxyurea treated
cells. The filters were sequentially probed with a probe specific
for HMG-17 mRNA and for Histone H4 mRNA.
early Gl; 18-hr, 94% mid-Gl; 22-hr, 86% Gl/S boundary and 14%
early-S. Total RNA from cells at different stages in the cell
cyle was isolated and examined either by Northern or by slot blot
analysis.
The autoradiogram presented in fig 5 compares the synthesis of
HMG-17 mRNA to that of histone H4 mRNA which is expressed during
the S phase of the cell cycle (13,24). HMG-17 transcripts are
present in the cell throughout the cell cycle however there is a
significant increase in HMG-17 mRNA 8 hr after release from the
double thymidine block, i.e during the late stage of S phase.
Fourteen hours after release the H4 mRNA is barely detectable
while that coding for HMG-17 is still present in considerable
amounts. Most of the HMG-17 transcription occurs during the S
phase of the cell cycle however, in contrast to histone transcription, HMG-17 transcription is not coupled to DNA synthesis.
As shown in fig 5B treatment with hydroxyurea, which stops both
DNA and H4 mRNA synthesis (24), does not affect the transcription
Of HMG-17 mRNA.
To obtain additional data, the RNA from all the time points was
slot blotted onto Zetabind and probed with nick translated
probes specific for either HMG-17 or H4. The autoradiogram of the
slot blot, clearly showed quantitative differences in the amount
of HMG-17 and H4 mRNA present in the various RNA samples. The
32
P counts bound to each slot were determined by excising the
slot and counting by liquid scintillation. The amount of each
probe bound to the RNA samples was plotted against the time after
release from the double thymidine block (Fig 6 ) . The probe
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A: HMG-17
o
X
g
CO
rx
UJ
a.
a.
O
0
4
8
12
16
HOURS AFTER RELEASE
Fig 6. Quantitation of cell cycle dependence of HMG-17 expression. Equal amounts of RNA isolated from cells at various stages
of the cell cycle were slot blotted on Zetabind filters, and
probed with nick translated probes specific for A, HMG-17; B,
Histone H4. The 32P counts bound to each of the slots was determined by liquid scintillation and plotted against the time of
release from the double thymidine block. The insert at the top of
A depicts the autoradiogram of the corresponsing slot blot.
specific for histone H4 indicated that the RNA isolated 4-8 hours
after release, i.e. during the S phase of the cell cycle, is
highly enriched in H4 mRNA. Examination of additional time
points reveals that the expression of the HMG gene seem to be
bimodal. A sharp increase in HMG mRNA level was observed early in
S, 2 hours after the release from the thymidine block. However,
the major wave of HMG-17 mRNA transcription was observed late in
S. The amount of HMG-17 mRNA remains stable through G2M and
declines in mid Gl.
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DISCUSSION.
The results presented indicate that, in human cells, the mRNA
coding for HMG-17 is very abundant. In HeLa cells this mRNA is 32
fold more abundant than the mRNA coding for actin and about 16
fold more abundant than the mRNA coding for histone H4. This
finding is unexpected since most cells contain more actin and H4
than HMG-17. Estimates of the molar ratio of H4 to HMG-17 range
from 4 to 100 (3). The results presented here suggests that the
low relative levels of cellular HMG-17 as compared to histones,
could be due to a more rapid turnover of the protein. Kuehl (25)
also noted that compared to histones, HMG-17 turns over at a
relatively fast rate however, Seale (26) reported that the HMG
proteins are not turning over at an unusually fast rate. The high
abundance of this mRNA is not a particular property of HeLa cells
since we have previously noted that A-549, HT-29 and MCF-7 cells
contain comparable levels of this RNA. In the human liver the
level of HMG-17 mRNA is only 15% of that found in HeLa (see fig
2C) however even at this level the amount of mRNA is significantly higher than that of most cellular messages coded by single
copy genes.
In human cells, HMG-17 is transcribed by one or more members of
a multigene family which has at least 50 gene copy equivalents.
The question arises as to the number of functionally active genes
coding for a functional transcript. Coir current studies (unpublished) suggest that some of the members of the HMG-17 multigene
families are processed retropseudogenes (27). The possible
relationship between mRNA levels and the occurence of
retropseudogenes has not yet been investigated. The high levels
of HMG-17 mRNA raises the possibility that the human cells
contain more than one functional gene. However it is also possible that the abundance of the message reflects a high transcript
stability due to the unusually long 3' untranslated region (69%
of the transcript). The possibile involvement of the transcript
terminals in the stability of the mRNA has been pointed out by
others (28).
Analysis of the HMG-17 mRNA levels at different stages of the
cell cycle reveals a sharp, temporal, increase in the mRNA levels
at the beginning of S-phase followed by a second wave of transcription which occurs late in S, while the synthesis of histone
H4 is still in progress (see fig 5 and 6 ) . It is possible that
the bimodality of the HMG-17 expression is due to transcription
of different genes at the various stages of the cell cycle. In
fact examination of Northern blots (for example see fig 5)
reveals that a certain amount of HMG-17 mRNA is present in the
cells throughout the cell cycle. This is most obvious when com-
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pared to the distinct cell cycle dependence of H4 mRNA which is
present only during S. In comparing the two stages of HMG-17
synthesis we note that most of the HMG-17 is transcribed in the
second stage, during late S, while both histone and DNA synthesis
are still in progress. However the synthesis of HMG-17 mRNA,
unlike that of the histone mRNAs, is not coupled to DNA synthesis
since inhibition of DNA synthesis does not affect HMG-17 mRNA
synthesis. The synthesis of HMG-17 protein seems to follow the
pattern of mRNA synthesis since it has been reported that incorporation of radioactivity into HMG-17 was greatest during DNA
replication (25) . Conceivably this is the stage at which HMG-17
is deposited on chromatin. The binding of the protein to newly
synthesized chromatin, is in agreement with the finding that
HMG-17 is a core particle, rather then DNA, binding protein
(2,6).
The amount of information on the transcriptional and
translational regulation of the entire class of HMG proteins is
very scarce (29) . We have previously noted that the mRNA for
HMG-1 and HMG-2 is polyadenylated (22) . Pentecost and Dixon (30)
reported that, in trout, the expression of the HMGl/2-like
proteins is not coupled to that of hlstones. The present manuscript, which is the first study concerning the transcriptional
regulation of HMG-17, indicates that elucidation of the regulatory processes of this gene may shed light on the cellular role of
the gene product. Further studies on the expression of the HMG-17
and HMG-14 during the various phases of the cell cycle and on the
stability of the message seem to be warranted.
Acknowledgements. We wish to thank Drs. G. and J. Stein for a
gift of plasmid plO8A, to Dr. N.Batulla for a gift of plasmid
pHA8H, Dr.W. Bonner for RNA from hydroxyurea treated cells, to
Dr. F. Gonzalez for rat RNA and to Dr D. Hatfield for critically
reviewing the manuscript.
REFERENCES.
T. Weintraub, H. and Groudine, M. (1976) Science 193, 848-856.
2. Weisbrod, S. (1982) Nature 297, 289-295.
3. Einck, L. and Bustin, M. (1985) Exp. Cell Res. 156, 295-310.
4. Yau, P., Imai, B.S., Thorne, A.W., Goodwin,G.H. and Bradbury,
E.M. (1983) Nucleic Acids Res 11, 2651-2664.
5. Mardian, J.K.W., Paton, A.E., Bunich, G.J. and Olins, D.E.
(1980) Science 209, 1534-1536
6. Sandeen, G. Wood, W.I. and Felsenfeld, G. (1980) Nucleic
Acids Res. 8, 3757-3578.
7. Shick, V.V., BelyavBky, A.V. and Mirzabekov, A.D. (1985) J.
Mol. Biol. 185, 329-339.
8.
3560
Einck, L. and Bustin M. (1983) Proc. Nat. Acad. S c i . 80,
6735-6739
Nucleic Acids Research
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Westermann, R. and Grossbach, U. (1984) Chromosoma 90,
355-365.
Dorbic, T. and Wittig, B. (1986) Nucleic Acid Res. 14,
3363-3376.
Druckmann, S., Mendelson, E., Landsman, D. and Bustin, M.
(1986) Exp. Cell Res. 166, 486-496.
Landsman, D., Soares, N., Gonzalez, F.J. and Bustin, M.
(1986) J. Biol. Chem. 261, 7479-7484.
Schumperli, D. (1986) Cell45, 471-472.
Foster, K.A. and Collins, J.M. (1985) J. Biol. Chem. 260,
4229-4235.
Maniatis, T. Fritsch, E.F. and Sambrook, J. "Molecular
Cloning;A Laboratry Manual" Cold Spring Harbor Laboratory
Publication 1982.
Aviv,H. and Leder, P. (1972) Proc. Nat. Acad. Sci. 69,
1408-1412.
Southern, E. (1975) J. Mol. Biol.98, 503-517.
Church, G. and Gilbert, W. (1984) Proc. Nat. Acad.Sci. 81,
1991-1995.
Landsman, D., Srikantha, T., Westermann, R. and Bustin, M.
(1986) J. Bio. Chem. 261, 16082-16086.
Sierra, F., Lictler, A.,Marashi, F., Rickles, R., Van Dyke,
T., Clark,S., Wells, J., Stein, G., and Stein, J. (1982)
Proc. Nat. Acad. Sci. 79, 1795-1800.
Berk, A.J. and Sharp, P.A. (1977) Cell 12, 721-732.
Bustin, M., Neihart N.K. and Fagan, J.B. (1981) Biochem.
Blophys. Res. Comm. 101, 893-897.
Johns, E.W.(ed)(1982) The HMG Chromosomal Proteins. Academic
Press, London.
Stein, G.S., Stein, J.L. and Marzluff, W.M. (ed)(1984)
Histone genes. John Willey and Sons, New York.
Kuehl,L. (1979) J. Biol. Chem. 254, 7276-7281.
Seale, L. R., Annunziato, A.T. and Smith, R.D. (1983)
Biochem. 22, 5008-5015.
Weiner, A. M., Deninger, P.L. and Estratiadis, A. (1986) Ann.
Rev. Biochem. 55, 631-661
Ross, J. and Kobs, G. (1986) J. Mol. Biol. 188, 579-593.
Smith, B.J.(1982) in Johns, E.W. (ed) The HMG Chromosomal
Proteins, Academic Press, plll-122.
Pentecost, B.T., Wright, J.M. and Dixon, G.H. (1985) Nucleic
Acids Res. 13, 4871-4888.
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