From the Medical Institute of Bioregulation, Kyushu
University, and CREST, Japan Science and Technology Corporation, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, the § Department of
Biochemical Genetics, Medical Research Institute, Tokyo Medical and
Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, and
the Departments of ¶ Chemistry and ** Urology, Faculty
of Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi
783-8505, Japan
Received for publication, February 3, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Elongin A is a transcription elongation factor
that increases the overall rate of mRNA chain elongation by RNA
polymerase II. To investigate the function of Elongin A in
vivo, the two alleles of the Elongin A gene have been disrupted
by homologous recombination in murine embryonic stem (ES) cells. The
Elongin A-deficient ES cells are viable, but show a slow growth
phenotype because they undergo a delayed mitosis. The cDNA
microarray and RNase protection assay using the wild-type and Elongin
A-deficient ES cells indicate that the expression of only a small
subset of genes is affected in the mutant cells. Taken together, our
results suggest that Elongin A regulates transcription of a subset but not all of genes and reveal a linkage between Elongin A function and
cell cycle progression.
Eukaryotic messenger RNA synthesis by RNA polymerase II (pol
II)1 is regulated by the
concerted action of a set of general transcription factors that control
the activity of pol II during the initiation and elongation stages of
transcription. At least six general transcription initiation factors
(TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) have been identified in
eukaryotic cells and found to promote the selective binding of pol II
to promoters and to support a basal level of transcription (1). In
addition to the general initiation factors, at least 17 elongation
factors have been defined biochemically and found to promote efficient
elongation of transcripts by pol II (2-5). These elongation factors
fall into two broad functional classes based on their abilities to
either reactivate arrested pol II or suppress transient pausing of pol
II. The first class is solely composed of SII (6). The rest of the
elongation factors, including TFIIF (7), Elongin A (8), Elongin A2 (9),
Elongin A3 (10), and Cockayne syndrome B (CSB) protein (11), all act to
increase the overall rate of mRNA chain elongation by decreasing
the frequency and/or duration of transient pausing of pol II at sites
along the DNA template.
Elongin was initially identified as a heterotrimer composed of A, B and
C subunits of ~770, 118, and 112 amino acids, respectively (8,
12-14). Elongin A is the transcriptionally active subunit of the
Elongin complex, whereas Elongins B and C are positive regulatory
subunits. Biochemical studies have shown that Elongins B and C form a
stable Elongin BC complex that binds to Elongin A and strongly induces
its transcriptional activity (8, 15). Elongin C functions as the
inducing ligand and activates transcription through interaction with a
short conserved motif (consensus sequence (T,
S)(LXXXCXXX)(V,L,I)) in the elongation activation
domain of Elongin A (15). Elongin B, a member of the ubiquitin homology protein family, appears to play a chaperone-like role in the assembly of the Elongin complex by binding to Elongin C and facilitating its
interaction with Elongin A (8, 14). Notably, Elongins B and C are also
found as integral components of a multisubunit complex containing the
von Hippel-Lindau (VHL) tumor suppressor protein (16, 17). Elongin A
and the VHL protein share the Elongin BC binding site motif; and
consistent with the assumption of a role for Elongin BC in tumor
suppression, more than 70% of VHL mutations found in VHL families and
sporadic clear cell renal carcinomas are associated with a mutation or
deletion at this site (16, 18).
To gain more insight into the role of Elongin A in vivo, we
have inactivated both alleles of the Elongin A gene in mouse ES cells.
We found that Elongin A is not essential for the viability of ES cells.
Though, surprisingly, such Elongin A-deficient ES cells display several
abnormalities, in cell size, cell growth, and cell cycle distribution,
which are similar to those of cells with a delayed onset of mitosis.
Furthermore, the absence of Elongin A protein affected the expression
of only a subset of class II genes, indicating that mammalian Elongin A
seemed to be required for pol II transcription of a subset but not all
of genes.
Generation of the Elongin A-deficient ES Cell Clones--
A
15-kb genomic fragment spanning the sequence encoding exons 2-11 and
the 3'-UTR of murine Elongin A was isolated from a 129/Sv mouse genomic
library, using a mouse Elongin A cDNA probe (19). Genomic fragments
of 4.5 and 1.4 kb flanking exons 8-10 of Elongin A were used to
generate a targeting construct in which exons 8-10 were replaced by a
pol II-neo-poly(A) cassette (20). To
increase the frequency of gene targeting, a pair of herpes simplex
virus (HSV)-1 and HSV-2 thymidine kinase (TK) cassettes (TK1 and TK2,
both under control of the MC1 promoter) was placed flanking the Elongin
A genomic sequence in the targeting vector (21, 22). CCE ES
cells were electroporated with the SalI-linearized targeting
vector as described previously (23, 24), and heterozygous Elongin
A-deficient clones were selected in the presence of 0.3 mg/ml G418
(Sigma) and 5 µM ganciclovir (Japan Syntechs). Among 200 ES cell colonies, three correctly targeted clones were identified by
Southern blot analysis, using 5' and 3' external probes located outside
the homologous regions of the targeting vector. Subsequently, homozygous Elongin A-deficient clones were obtained by selection in
higher concentrations of G418 (from 1.5 to 2.0 mg/ml).
Southern Blot Analysis--
Genomic DNA was isolated from ES
cells, digested with either XbaI or BssHII,
separated on a 0.65% agarose gel, and transferred to nylon membranes
(Amersham Biosciences) by capillary blotting. The membrane was
hybridized with random-primed 32P-labeled external probe
(Fig. 1A) by using standard methods.
RT-PCR Analysis--
Total RNA was prepared from cultured cells
using ISOGEN (Nippon Gene). First-strand cDNA was synthesized using
a first-strand cDNA synthesis kit (Amersham Biosciences) as
described in the manufacturer's instructions. Subsequently, PCR was
performed with primers A1 (5'-GCGTCCCAGAGATGCTCTTCAG-3') and A2
(5'-TTATCGCCGGGAGAATCTGTTCTTGAATG-3') to confirm the deletion of
Elongin A. RT-PCR analysis of GAPDH mRNA was also performed as a
control with primers mGA1 (5'-CTGCCATTTGCAGTGGCAAAG-3') and mGA2
(5'-TGGTATTCAAGAGAGTAGGGA-3').
Cell Proliferation Assay--
In vitro cell growth
was assessed by plating 1 × 104 cells per well in
six-well tissue culture dishes in triplicate. Cells were harvested at
24-h intervals by trypsinization and counted using a hemocytometer.
Fluorescence-activated Cell Sorting (FACS) Analysis--
FACS
analysis was performed as described previously (25, 26). Briefly, ES
cells were harvested and fixed with 70% ethanol. After being washed
with ice-cold phosphate-buffered saline, fixed cells were stained for
cellular DNA with propidium iodide (Sigma). The cell suspension was
passed through a nylon mesh membrane and DNA content and cell numbers
were analyzed with a FACS Calibur (Becton Dickinson). For each sample,
1 × 104 cells were analyzed, and the results were
processed through Cell Quest software (BD Biosciences).
Cytogenetic Analysis--
Chromosome slides were prepared
according to the standard cytogenetic method. Briefly, cultured cells
were treated with 1.5 µg/ml TN-16
(3-(1-anilinoethylidene)-5-benzylpyrolidine-2,4-dione, Wako) for 2 h to collect metaphase cells. Cells were harvested using 0.25%
trypsin-EDTA and centrifuged at 1000 rpm for 5 min. Cells were treated
with hypotonic solution (75 mM KCl) for 20 min at room
temperature and fixed with Carnoy's fixative (methanol:acetic acid = 3:1). Cell suspensions were dropped onto slide glass, then air-dried. Chromosome slides were stained with 3% Giemsa in
phosphate-buffered saline for 10-20 min. Chromosome numbers were
evaluated using a Zeiss Axiophot microscope under a ×100 objective.
Microarray Analysis--
Poly(A)+ RNA was isolated
according to the manufacturer's protocol using a MACS mRNA
isolation kit (Miltenyi Biotec). cDNA generation,
hybridization, and data collection were performed by Incyte Genomics.
In brief, alterations in gene expression were evaluated by reverse
transcription of poly(A)+ RNAs in the presence of Cy3 or
Cy5 fluorescent labeling dyes followed by hybridization to a mouse GEM
2 microarray chip. Each chip carries a total of 9509 cDNAs. The
data were analyzed using GEM Tools 2.5 software (Incyte
Pharmaceuticals, Inc.).
RNase Protection Assay--
The RNase protection assay was
performed using the mouse cell cycle regulator multiprobe template
sets, mCYC-1, mCYC-2, and mCC-1 (BD Biosciences).
[32P]UTP-labeled antisense RNA probe was synthesized
using T7 RNA polymerase and was purified by phenol-chloroform
extractions and ethanol precipitation. Purified probe was mixed with 10 µg of total RNA from ES cells in hybridization buffer and incubated for 16 h at 56 °C. Free probe and single-stranded RNA molecules were digested with a mixture of RNase A and T1. The RNase-protected molecules were purified and resolved on a denaturing polyacrylamide gel
and dried. Autoradiographic signal was scanned on a BAS2000 image
analyzer (Fuji), and the signal intensity of each band was quantified
using MacBAS software (Fuji) and normalized to the corresponding GAPDH levels.
To understand the physiological role of Elongin A in
vivo, we disrupted the Elongin A gene in mouse ES cells. We first
isolated and analyzed the structure of the mouse Elongin A gene (19). A
gene-targeting vector was then constructed by replacing exons 8-10 of
the Elongin A gene, which encode the domain essential for its
transcriptional elongation activity (15), with a pol II-neo-poly(A) cassette (Fig.
1A). Three independent
heterozygous Elongin A-deficient ES cell lines were obtained by
homologous recombination, and subsequently, homozygous Elongin
A-deficient ES cell lines were generated by culturing these
heterozygous ES cells in higher concentrations of G418. Genotyping of
the isolated clones was determined by Southern blot analysis with
flanking 5'- and 3'-UTR probes (Fig. 1B) and confirmed by
RT-PCR (Fig. 1C).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Generation of Elongin A-deficient ES cell
clones. A, diagrams summarizing the genomic region at
the Elongin A locus, the Elongin A targeting vector, and the predicted
structure of the mutated Elongin A gene. The coding exons are depicted
by closed boxes and are numbered. The direction of Elongin A
gene transcription is left to right, and the
arrow indicates the direction of the Neo cassette. TK1 and
TK2 are the herpes simplex virus type 1 and type 2 TK genes,
respectively. The 5' and 3' probes used for Southern hybridization are
indicated. A1 and A2 are the oligonucleotides used for RT-PCR
analysis. B, Southern blot analysis of genomic DNA purified
from the wild-type (+/+), heterozygous Elongin A-deficient (+/ ), and
homozygous Elongin A-deficient (
/
) ES cell clones. XbaI-
or BssHII-digested genomic DNA was hybridized with the 5' or
3' probe. Fragments of 3.2 and 5.3 kb in the left panel, 37 and 23 kb in the middle panel, and 37 and 15 kb in the
right panel represent wild-type allele and targeted allele,
respectively. C, RT-PCR analysis of mRNA from the
wild-type (+/+), heterozygous Elongin A-deficient (+/
), and
homozygous Elongin A-deficient (
/
) ES cell clones. The primer set
(A1/A2) directs the amplification of a 2034-bp fragment from wild-type
allele-derived cDNA. RT-PCR analysis of GAPDH mRNA was also
performed as a control.
To investigate the phenotype of the Elongin A-deficient ES cells, we
first compared growth rates of Elongin A-deficient and wild-type ES
cells in vitro. As shown in Fig.
2A, homozygous Elongin A-deficient ES cells grew about three times as slowly as wild type ES
cells, whereas heterozygous Elongin A-deficient ES cells grew at rates
indistinguishable from those of wild-type cells. Moreover, microscopic
analysis revealed that the cellular and nuclear sizes of homozygous
Elongin A-deficient ES cells were significantly larger than those of
wild type and heterozygous deficient ES cells (Fig. 2B and
data not shown).
|
To clarify the cause of the growth defects of the homozygous Elongin
A-deficient ES cells, we first used FACS analysis to determine cell
cycle profiles. In contrast to wild type and heterozygous Elongin
A-deficient ES cells, significantly more G2/M phase cells (or cells with a DNA content of 4N) than G1 phase cells
were present in the homozygous Elongin A-deficient population. We also
observed that homozygous Elongin A-deficient ES cells, but none of the other cells, included a substantial fraction with a DNA content of 8N
(Fig. 3A). The increase of
cells with a DNA content of 4N and the appearance of cells with that of
8N in the homozygous Elongin A-deficient population were confirmed by
cytogenetic analysis (Fig. 3, B and C). These
results imply that the G2/M phase is prolonged in
homozygous Elongin A-deficient ES cells and that homozygous Elongin
A-deficient ES cells go through DNA rereplication, even after
completion of one cycle or DNA replication.
|
Together, the observed abnormalities in homozygous Elongin A-deficient ES cells, such as increased cell size, more G2/M phase cells, and the presence of products of DNA rereplication, correspond to those of cells with delayed mitosis, which can be caused either by inhibition of G2/M transition or by stimulation of the initiation of DNA replication.
We next intended to determine the mechanism responsible for the
observed delayed mitosis. Thus, a cDNA microarray analysis was
applied to compare populations and levels of mRNA in ES cells carrying or lacking functional Elongin A. Wild-type or homozygous Elongin A-deficient ES cells were grown to log phase, then
poly(A)+ RNA was isolated and used for cDNA synthesis
and subsequently hybridized to a microarray chip. Under our
experimental conditions, mRNAs whose levels were decreased or
increased by 2-fold or greater were considered as differentially
expressed transcripts. The expression of the vast majority of genes was
unaffected by disruption of the Elongin A gene. However, 241 (2.5%) of
the genes assayed had a significantly down-regulated expression in
Elongin A-deficient ES cells. Interestingly, a roughly equivalent
number of genes was up-regulated by Elongin A inactivation (Fig.
4A and see Supplemental Tables). However, we presently do not know whether this is a
consequence of secondary effects due to loss of Elongin A function as a
transcription elongation factor or primary effects caused by loss of
Elongin A function, which repress the expression of a subset of
genes.
|
To verify the results of the microarray analysis and also to identify
genes responsible for the delayed mitosis phenotype, an RNase
protection assay was carried out for quantitative estimation of the
expression of cell cycle-related genes in ES cells (Fig. 4B
and data not shown). Consistent with the microarray analysis, cyclin E
mRNA, but not CDK2, cyclin D2, and cyclin D3 mRNAs, were down-regulated by inactivation of Elongin A. However, the expression of
the other tested cell cycle-related genes, including the mRNAs of
key regulators of G2/M transition, such as cyclin A2,
cyclin B1, cyclin B2, and cdc2, was either not, or was only weakly,
altered in Elongin A-deficient ES cells.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extensive study in vitro has demonstrated a requirement for transcription elongation factors in transcriptional regulation; however, the functions of these factors in vivo have not been properly evaluated. Here we report that mammalian cells in which a gene encoding one of the transcription elongation factors, Elongin A, was disrupted by homologous recombination had (i) a delayed mitosis phenotype and (ii) no general, but rather specific, defects in RNA polymerase II-directed transcription.
Although we have not been able to specify how a lack of Elongin A leads to delayed mitosis, we can assume several possible mechanisms. One possibility is that Elongin A is required for the transcription of specific genes involved in G2/M transition, and a reduction in those transcripts caused by Elongin A inactivation results in a delayed onset of mitosis. In this scenario, Elongin A could be a target for gene-specific transcriptional activators and recruited to the promoter regions of specific genes. Indeed, both a gene-selective action and delayed mitosis phenotype following inactivation of general transcription factors have been reported (27-30). TATA-binding protein (TBP) is critical for the transcription of cdc25B (cdc2 phosphatase), and the reduction of cdc25B expression by heterozygous deletion of TBP causes the accumulation of hyperphosphorylated, inactive cdc2, which in turn leads to delayed mitosis (27). Furthermore, human TBP-associated factor 150 (TAF150) or yeast TAF90 is also specifically important for the transcription of cyclin B1 and cyclin A, or SPC98 (a component of the spindle pole body) and APC2 (a component of the anaphase-promoting complex), respectively, and its deletion results in G2/M cell cycle arrest (28, 30). Thus, it is possible that Elongin A is required for one or more genes involved in progression through the G2/M boundary of the cell cycle, although our initial study has not yet identified such genes. The other possibility is that there might be a functional link between transcription elongation by Elongin A and DNA recombination, as was found in Saccharomyces cerevisiae transcription elongation factors, HPR1 and THO2 (31-33). Mutations in these two yeast genes have been known to induce a hyper-recombination phenotype, which perhaps results from recruitment of the recombination machinery to the vicinity of inappropriately paused RNA polymerase II elongation complexes. If this is also the case in mammalian Elongin A, lack of functional Elongin A may induce a hyper-recombination phenotype and genomic instability, which could result in mitosis being delayed by the G2 checkpoint mechanism, although we presently do not have direct evidence of physical interaction between Elongin A and recombination proteins. However, it is also possible that Elongin A carries out some other cellular functions unrelated to transcription elongation in vivo. In fact, Elongin A has recently been shown to form a stable complex with the known components of the ubiquitin ligase, Cul5 and Rbx1 (34). Although we presently do not know whether this complex actually possesses ubiquitin ligase activity, an intriguing possibility is that Elongin A together with Cul5 and Rbx1 mediate the degradation of factors, such as inhibitors of the mitosis promoting factor and positive regulators of the initiation of DNA replication, by a ubiquitin-proteasome pathway. Thus, Elongin A may function in the cell cycle by a mechanism that does not operate directly through a transcription pathway.
In summary, our results demonstrate that mammalian Elongin A is
essential for transcription of a subset but not all of genes and is
important for proper cell cycle progression. Further in vitro experiments will be necessary to understand the precise molecular mechanisms by which loss of functional Elongin A results in
delayed onset of mitosis.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the National Space Development Agency of Japan; and the CREST.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary Tables S1 and S2.
To whom correspondence may be addressed: Dept. of Chemistry,
Faculty of Medicine, Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan. Tel.: 81-88-880-2279; Fax: 81-88-880-2281; E-mail: asot@kochi-ms.ac.jp (for T. A.) or Dept. of Biochemical Genetics, Medical Research Inst., Tokyo Medical & Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel.:
81-3-5803-5822; Fax: 81-3-5803-0248; E-mail: kita.bgen@mri.tmd.ac.jp
(for S. K.).
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.C300047200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pol II, RNA polymerase II; ES, embryonic stem; TF, transcription factor; VHL, von Hippel-Lindau; neo, neomycin resistance gene; HSV, herpes simplex virus; TK, thymidine kinase; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FACS, fluorescence-activated cell sorting; TBP, TATA-binding protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Roeder, R. G. (1996) Trends Biochem. Sci. 21, 327-335[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Aso, T.,
Conaway, J. W.,
and Conaway, R. C.
(1995)
FASEB J.
9,
1419-1428 |
3. | Reines, D., Conaway, J. W., and Conaway, R. C. (1996) Trends Biochem. Sci. 21, 351-355[CrossRef][Medline] [Order article via Infotrieve] |
4. | Uptain, S. M., Kane, C. M., and Chamberlin, M. J. (1997) Annu. Rev. Biochem. 66, 117-172[CrossRef][Medline] [Order article via Infotrieve] |
5. | Conaway, J. W., Shilatifard, A., Dvir, A., and Conaway, R. C. (2000) Trends Biochem. Sci. 25, 375-380[CrossRef][Medline] [Order article via Infotrieve] |
6. | Wind, M., and Reines, D. (2000) Bioessays 22, 327-336[CrossRef][Medline] [Order article via Infotrieve] |
7. | Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1989) Mol. Cell. Biol. 9, 1465-1475[Medline] [Order article via Infotrieve] |
8. | Aso, T., Lane, W. S., Conaway, J. W., and Conaway, R. C. (1995) Science 269, 1439-1443[Medline] [Order article via Infotrieve] |
9. |
Aso, T.,
Yamazaki, K.,
Amimoto, K.,
Kuroiwa, A.,
Higashi, H.,
Matsuda, Y.,
Kitajima, S.,
and Hatakeyama, M.
(2000)
J. Biol. Chem.
275,
6546-6552 |
10. |
Yamazaki, K.,
Guo, L.,
Sugahara, K.,
Zhang, C.,
Enzan, H.,
Nakabeppu, Y.,
Kitajima, S.,
and Aso, T.
(2002)
J. Biol. Chem.
277,
26444-26451 |
11. |
Selby, C. P.,
and Sancar, A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11205-11209 |
12. |
Bradsher, J. N.,
Jackson, K. W.,
Conaway, R. C.,
and Conaway, J. W.
(1993)
J. Biol. Chem.
268,
25587-25593 |
13. | Garrett, K. P., Tan, S., Bradsher, J. N., Lane, W. S., Conaway, J. W., and Conaway, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5237-5241[Abstract] |
14. | Garrett, K. P., Aso, T., Bradsher, J. N., Foundling, S. I., Lane, W. S., Conaway, R. C., and Conaway, J. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7172-7176[Abstract] |
15. | Aso, T., Haque, D., Barstead, R. J., Conaway, R. C., and Conaway, J. W. (1996) EMBO J. 15, 5557-5566[Abstract] |
16. | Duan, D. R., Pause, A., Burgess, W. H., Aso, T., Chen, D. Y. T., Garrett, K. P., Conaway, R. C., Conaway, J. W., Linehan, W. M., and Klausner, R. D. (1995) Science 269, 1402-1406[Medline] [Order article via Infotrieve] |
17. | Kibel, A., Iliopoulos, O., DeCaprio, J. A., and Kaelin, W. G. (1995) Science 269, 1444-1446[Medline] [Order article via Infotrieve] |
18. | Kishida, T., Stackhouse, T. M., Chen, F., Lerman, M. I., and Zbar, B. (1995) Cancer Res. 55, 4544-4548[Abstract] |
19. | Aso, T., Amimoto, K., Takebayashi, S., Okumura, K., and Hatakeyama, M. (1999) Cytogenet. Cell Genet. 86, 259-262[CrossRef][Medline] [Order article via Infotrieve] |
20. | Deng, C., Thomas, K. R., and Capecchi, M. R. (1993) Mol. Cell. Biol. 13, 2134-2140[Abstract] |
21. | Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Nature 336, 348-352[CrossRef][Medline] [Order article via Infotrieve] |
22. | Rancourt, E. D., Tsuzuki, T., and Capecchi, M. R. (1995) Genes Dev. 9, 108-122[Abstract] |
23. | Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E. J., ed) , IRL Press, NY |
24. | Joyner, A. L. (1993) in Gene Targeting: A Practical Approach (Joyner, A. L., ed) , IRL Press, NY |
25. | Oda, S., Nishida, J., Nakabeppu, Y., and Sekiguchi, M. (1995) Oncogene 10, 1343-1351[Medline] [Order article via Infotrieve] |
26. | Tominaga, Y., Tsuzuki, T., Shiraishi, A., Kawate, H., and Sekiguchi, M. (1997) Carcinogenesis 18, 889-896[Abstract] |
27. |
Um, M.,
Yamauchi, J.,
Kato, S.,
and Manley, J. L.
(2001)
Mol. Cell. Biol.
21,
2435-2448 |
28. |
Martin, J.,
Halenbeck, R.,
and Kaufmann, J.
(1999)
Mol. Cell. Biol.
19,
5548-5556 |
29. | Apone, L. M., Virbasius, C. A., Reese, J. C., and Green, M. R. (1996) Genes Dev. 10, 2368-2380[Abstract] |
30. | Lee, T. I., Causton, H. C., Holstege, F. C. P., Shen, W., Hannett, N., Jennings, E. G., Winston, F., Green, M. R., and Young, R. A. (2000) Nature 405, 701-704[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Chavez, S.,
and Aguilera, A.
(1997)
Genes Dev.
11,
3459-3470 |
32. |
Piruat, J. I.,
and Aguilera, A.
(1998)
EMBO J.
17,
4859-4872 |
33. | Chavez, S., Beilharz, T., Rondon, A. G., Erdjument-Bromage, H., Tempst, P., Svejstrup, J. Q., Lithgow, T., and Aguilera, A. (2000) EMBO J. 21, 5824-5834 |
34. |
Kamura, T.,
Burian, D.,
Yan, Q.,
Schmidt, S. L.,
Lane, W. S.,
Querido, E.,
Branton, P. E.,
Shilatifard, A.,
Conaway, R. C.,
and Conaway, J. W.
(2001)
J. Biol. Chem.
276,
29748-29753 |