Long Telomeric C-rich 5'-Tails in Human Replicating Cells*
Graziella
Cimino-Reale
,
Esterina
Pascale§,
Ester
Alvino
,
Giuseppe
Starace
, and
Ettore
D'Ambrosio
¶
From the
Istituto di Neurobiologia e Medicina
Molecolare, Consiglio Nazionale delle Ricerche, 00137 Roma, Italy,
§ Dipartimento di Medicina Sperimentale e Patologia,
Università "La Sapienza," 00185 Roma, Italy
Received for publication, September 3, 2002, and in revised form, October 24, 2002
 |
ABSTRACT |
Telomeres protect the ends of linear chromosomes
from abnormal recombination events and buffer them against terminal DNA
loss. Models of telomere replication predict that two daughter
molecules have one end that is blunt, the product of leading-strand
synthesis, and one end with a short G-rich 3'-overhang. However,
experimental data from proliferating cells are not completely
consistent with this model. For example, telomeres of human chromosomes
have long G-rich 3'-overhangs, and the persistence of blunt ends is
uncertain. Here we show that the product of leading-strand synthesis is
not always blunt but can contain a long C-rich 5'-tail, the
incompletely replicated template of the leading strand. We examined the
presence of G-rich and C-rich single-strand DNA in fibroblasts and HeLa cells. Although there were no significant changes in the length distribution of the 3'-overhang, the 5'-overhangs were mostly present
in S phase. Similar results were obtained using telomerase-negative fibroblasts. The amount and the length distribution of the 5' C-rich
tails strongly correlate with the proliferative rate of the cell
cultures. Our results suggest that, contrary to what has commonly been
supposed, completion of leading-strand synthesis is inefficient and
could well drive telomere shortening.
 |
INTRODUCTION |
Telomeres are complex specialized structures that seal the termini
of eukaryotic chromosomes protecting them from DNA loss, end-to-end
fusion, and other potential genetic rearrangements. Telomeric DNA of
most eukaryotic organisms is made of short tandem repeated sequences
(1-4). The nature and the organization of the repeats is such that one
strand is usually rich in guanines (G), and this strand always runs 5'
to 3' toward the end of the chromosome. Consequently, its complementary
cytosine (C)-rich strand always runs 5' to 3' toward the centromere.
When a telomere is replicated, leading-strand synthesis produces the
G-rich copy of the C-rich template terminus, and lagging-strand
synthesis produces the C-rich copy of the G-rich template terminus. As
a consequence, a terminal G-rich 3'-overhang at least as long as the
removed RNA primer from the parental strand is expected to be present
in the lagging-strand telomere, whereas the leading-strand telomere is
expected to be blunt (5, 6). Upon successive rounds of DNA replication,
telomeres will progressively shorten and, eventually all telomere
functions will be lost and the chromosomes will become unstable (7, 8).
However, a compensatory mechanism is provided by telomerase, which adds
telomeric repeats onto the 3' DNA end of chromosomes (9-12).
Although the exact structure of the 3'-overhang varies between species,
the presence of such overhangs is both conserved and believed to be
essential for maintenance of chromosome end structure and function. In
fact, a 3'-end of at least six nucleotides together with its associated
telomere-binding factors is thought to participate directly in forming
a specialized telomere structure (13, 14) (e.g. a
"t-loop"). In human cells, chromosome ends with long G-rich 3'-overhangs that vary from 100 to 280 nucleotides have been detected (15, 16). In these cells, the rate of telomere shortening during cell
doublings depends on the size of the overhang (17). In mammals, RNA
priming events are thought to occur about every 100-200 bp during
lagging-strand synthesis. This is roughly consistent with the rates of
telomere shortening of 40-200 bp per cell division that has been
observed in cultured human cells (11, 18). A possible explanation is
that human cells lack the ability to position the final RNA primer at
the very end of the chromosome. Several lines of evidence suggest that
the normal DNA replication machinery generates the C-rich complement of
the telomerase extended G-strand (4, 19) and that telomeric G-
and C-strand syntheses are coordinately regulated (20, 21). Because
telomerase requires a 3'-overhang, current models suggest that both
chromosomal termini should contain G-rich 3'-tails. Evidence has been
provided that Tetrahymena thermophila rDNA molecules have
overhangs on both telomeres (22). Consequently, the blunt termini that
have been generated by leading-strand synthesis would need to be
processed by a 5' to 3' exonuclease to erode the C-rich parental
template (23, 24-27), although this remains controversial (15,
16, 28).
We have recently developed a novel protocol, which we call
T-OLA,1 to examine the nature
of the chromosomal DNA termini in cell cultures and tissues (29). With
this method, we found quite unexpectedly that the 5' C-rich end of the
leading-strand template remains for a time unreplicated in
proliferating cells, e.g. it persists as a 5' C-rich tail
during S phase. This result suggests that the replication fork may
stall before reaching the very end of the chromosome. This in turn
could lead to excessive shortening during replication of the lagging
strand because the ability to position the RNA primer near the 3'-end
of the lagging G-rich parental strand would be compromised. This
scenario would account for both the long G-rich tails and the
accelerated telomere shortening thought to accompany them. In addition,
if the uncopied 5' C-rich tail gets eroded by the process that normally
exposes the G-rich tail on fully replicated leading-strand telomeres,
then telomere shortening would also be further enhanced.
 |
EXPERIMENTAL PROCEDURES |
Cell Cultures and DNA Extraction--
High molecular weight DNA
(30) was obtained from human foreskin fibroblasts cultured in Eagle's
basal medium plus 10% fetal calf serum and from HeLa cells
grown in Dulbecco's modified Eagle's medium plus 10% fetal calf
serum. HeLa cells were halted at the G1/S boundary by a
double treatment with 2 mM thymidine. Cells were harvested
0 and 4 h after the release. Resting fibroblasts were obtained
either by serum starvation or by harvesting the cells that were
maintained for at least 48 h in low serum medium (2%) after they
reached confluence. Cell starvation was produced by seeding 5 × 105 cells in a 175-cm2 flask with 30 ml of 10%
fetal calf serum Eagle's basal medium. Proliferating fibroblasts were
harvested from 50% confluent flasks. Synchrony was monitored by flow
cytometric measurements of DNA distribution. Typically, the
proliferating HeLa and fibroblast cell cultures presented respectively
75% and 40% of cell population in S phase.
Telomeric Oligonucleotide Ligation Assay--
T-OLA was
performed as previously described with minor modifications (29).
Briefly, oligonucleotides were phosphorylated by T4 polynucleotide
kinase. A mixture of 30 µl containing 5 pmol of oligonucleotide and
50 pmol of [
-32P]ATP (3000 Ci/mmol, 10 mCi/ml), 70 mM Tris, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, and 20 units of T4 polynucleotide kinase was incubated for 40 min at 37 °C. 0.3 µl of 0.1 M
unlabeled ATP and a further 10 units of kinase were then added, and the
reaction was continued for 15 min. Labeled oligonucleotides,
[(CCCTAA)2] and [(TTAGGG)2], were sodium
acetate/ethanol-precipitated and dissolved in an appropriate volume of
water. Hybridization and ligation were conducted in a volume of 20 µl
containing 5 µg of undenatured DNA, 0.5 pmol of oligonucleotide
probe, 50 mM Tris-HCl pH 7.5, 10 mM
MgCl2, 10 mM dithiothreitol, 1 mM
ATP, 25 µg/ml bovine serum albumin. All ingredients (except ligase)
were placed into 0.5-ml PCR tubes and incubated at 33 °C for 12-14
h. Subsequently, 150 units of T4 DNA ligase was added, and the reaction
was extended for a further 4 h. Reactions were ended by adding 30 µl of water and by phenol/chloroform extraction. Samples were
precipitated with ethanol and dissolved in 6 µl of 10 mM
Tris-HCl, pH 8.0, 1 mM EDTA.
Electrophoresis Analysis of Reaction Products--
Reaction
products were analyzed on denaturing 6% acrylamide sequencing gels. In
addition, reaction samples were resolved on non-denaturating 1%
agarose gels to check for DNA quantity, relationship and consistency
between observed hybridization signal to high molecular weight DNA and
T-OLA products. For analysis of ligated products under denaturing
conditions, half of the volume was mixed with 4 µl of formamide stop
solution. Samples were heated at 90 °C and immediately quenched on
ice before loading 2 µl onto the gel. The remaining 3 µl were
diluted in water and in a glycerol loading buffer to a final volume of
10 µl and run on a non-denaturing 1% agarose gel for 4 h at 90 V in Tris-acetate buffer. The gels were dried on nylon membrane
(Stratagene) and exposed to autoradiography film. The images were
acquired by 1D Image Analysis Software (Eastman Kodak Co.). Because the
intensity of each band depends on both the frequency of the specific
tail and on its length, the intensity (background subtracted) of each
band of the ladder was divided by the number of concatenated
oligonucleotide probes expected to be in the band so that the resulting
relative intensities would be proportional to the relative tail
frequency. This value was then normalized to the total intensity and
plotted both as relative frequency and as length distribution of the tails.
Enzymatic DNA Modification--
To remove the 3'-single-stranded
DNA, Escherichia coli Exonuclease I was used. DNAs were
incubated in 10 mM Tris HCl, pH 8.0, 1 mM EDTA,
10 mM MgCl2, 20 mM KCl, and 10 mM 2-mercaptoethanol and 1 unit/µl of enzyme Exonuclease
I (U. S. Biochemical Corp.) for 24 h at 37 °C. For cleaving
3'-recessed but not 3'-protruding ends of DNA, Exonuclease III was
used. DNA was incubated in 66 mM Tris HCl, pH 8.0, 6.6 mM MgCl2, 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, and 1 unit/µl Exonuclease III at
37 °C for 30 min. To remove the single-stranded regions, both in 5'
3' and 3'
5' directions, the DNA was incubated with 0.2 unit/µl Exonuclease VII (U. S. Biochemical Corp.) in 50 mM Tris HCl pH 7.9, 50 mM potassium phosphate
pH 7.6, 8.3 mM EDTA, and 10 mM 2-mercaptoethanol at 37 °C for 1 h. To remove the 5'
single-stranded DNA, the DNA was incubated with T7 (Gene6) Exonuclease
(0.6 unit/µl) (U. S. Biochemical Corp.) in 50 mM Tris
HCl pH 8.1, 5 mM MgCl2, 20 mM KCl,
and 5 mM 2-mercaptoethanol at 37 °C for 20 min. The reactions were stopped, the mixtures were extracted with
phenol-chloroform, and the DNA was precipitated and dissolved in
H2O.
 |
RESULTS |
In the OLA, two oligonucleotides are designed to hybridize
in exact juxtaposition to the target DNA sequences, permitting their
covalent joining by a DNA ligase. Because human telomeres are composed
of repetitions of a 6-nucleotide sequence, in frame hybridized
complementary oligonucleotide ligation of a telomeric sequence is
expected to produce a ladder of concatenated oligonucleotides whose
lengths are comparable with the target sequences. If single-stranded telomeric regions are present in native DNA, they will be replenished by in-frame oligonucleotides that will be covalently joined by ligase.
Our experimental approach for measuring the length of telomeric
overhangs is based on the ligation of oligonucleotides to undenatured
DNA using oligonucleotides designed to hybridize at high stringency,
and in frame, with telomeric repeats (T-OLA). The sensitivity and
specificity of this method have been extensively evaluated in a
previous report (29) in which the 3'-overhang length distribution of
various human cell cultures was reported. In the process of analyzing
the length of the 3'-overhang of various mammalian cell lines and
tissues, we unexpectedly detected in some cell cultures a ladder of
products also when an oligonucleotide complementary to the C-rich
strand was used. Although the length distribution of the
single-stranded segments was very similar to that obtained probing for
the G-rich strand, the total amount was variable but persistently
lower, ranging from 1/3 to 1/10 (absent in some tissues). However, the
total amount of C-rich single-stranded DNA appeared to correlate with
the proliferative status of the tested cells, e.g. it was
higher in fast proliferating cells. The results obtained with HeLa
cells, foreskin human fibroblasts, and circulating leukocytes are shown
in Fig. 1.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 1.
Detection of G-rich and C-rich
overhangs. The upper part of the panel shows
the ladder of products obtained on denaturing acrylamide gels after
T-OLA probing for the G-strand [(AATCCC)2] and for
C-strand [(TTAGGG)2] in HeLa cells (lanes 1),
in circulating leukocytes (lanes 2), and in foreskin
fibroblasts (lanes 3). One-half of the reaction was also
analyzed by native agarose gel electrophoresis. The middle
part of the panel shows the total amount of probe bound to high
molecular weight DNA. Ethidium Bromide staining of the gel is also
shown (lower).
|
|
Because the G-rich telomeric strand is always synthesized as the
leading strand, incomplete leading-strand replication should leave a 5'
C-rich overhang. The nature of the single-stranded DNA was investigated
by treating the DNA with some specific exonucleases. Enzymatic
treatment of DNA prior to T-OLA enables a series of predictions to be
tested. A 3'-tail is expected to be specifically digested by E. coli Exonuclease I but will be insensitive to Exonuclease III. Conversely, a 5'-tail will be insensitive to Exonuclease I
treatment but will be augmented by Exonuclease III. A 5'-exonuclease such as T7 (Gene 6) Exonuclease will increase the amount of
3'-overhang, lengthen the G-rich tail, and remove the 5'-overhang.
Exonuclease VII, which is a strict single-strand directed enzyme with
5' to 3' and 3' to 5' exonuclease activity, will be able to distinguish tails by internal gap of the double helix. The predicted and observed effect of nuclease treatment of DNA before performing T-OLA with C-strand- and G-strand-specific oligonucleotides confirmed that the
5'-overhang was the telomeric C-rich tail (Fig.
2). The partial resistance of the G
strand to the Exonuclease I and Exonuclease VII activity suggests also
that some secondary structure of the telomere (e.g. a
"t-loop") may be present.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of nuclease treatments on the T-OLA
reaction. A, predicted effects of enzymatic treatments on
T-OLA. The enzymatic effects able to discriminate between 5'-tail
(a) or 3'-tail (b), and internal gaps are
indicated. B, observed effects of enzymatic treatments.
Native, undigested DNA; ExoI, Exonuclease I; 5'Exo, T7 (Gene6)
Exonuclease; Exo VII, Exonuclease VII; Exo III, Exonuclease III. The
results obtained from enzymatic treatments of DNA extracted from
proliferating cells (in this case HeLa cells) are shown. Compare
quantity and quality of the ladder products with untreated DNA. The
predicted and observed effects are consistent with 3' G-rich and 5'
C-rich overhangs.
|
|
We then examined the nature of the DNA termini in HeLa cell lines and
in human foreskin fibroblasts, cells that lack detectable telomerase
activity. For this purpose, we blocked HeLa cells by double thymidine
shock. Samples were analyzed at the G1/S boundary and at
full S phase (75% of the cell in S phase). Samples from resting
fibroblasts, obtained either by cell starvation or by maintaining
confluent monolayers in low serum, were compared with samples derived
from high proliferating fibroblasts (40% of cells in S phase). The
results are shown in Fig. 3. Comparable
amounts and lengths of 3' G-rich tails are present in the two
proliferative conditions both of HeLa and fibroblasts, albeit with
somewhat higher amounts in S phase. In contrast, substantial amounts of the 5' C-strand tail are present only in S phase. In addition, far more
of the 5' C-strand tails are skewed toward longer lengths than the G-
strand. Fig. 3 also shows that very long G-rich and C-rich tails are
present during the log phase of proliferating cells. In contrast, the
amount and length of the G-rich tails are slightly reduced in resting
cells, whereas the C-rich tails are virtually absent.

View larger version (76K):
[in this window]
[in a new window]
|
Fig. 3.
G-rich 3'- and C-rich 5'-overhangs in resting
and proliferating HeLa and fibroblasts. A, T-OLA products.
The G- and the C-strand ladders are obtained by probing HeLa cell DNA
in G1/S and S phases, respectively with
[(AATCCC)2] and [(TTAGGG)2]
oligonucleotides. Resting (R) and proliferating fibroblast
cells (P) were also studied. B, comparison of the
quantity and quality of the tails. The histograms are deduced by
quantitative analysis carried out with 1D Image analysis software
(Kodak). The relative amounts and the length distributions of the G-
and the C-tails in the two cell cycle phases are plotted as relative
intensity. The lighter patterns indicate the
percentage of tails shorter than 90 nucleotides, the dark
part the percentage of tails longer than 90 nt.
|
|
 |
DISCUSSION |
We show for the first time that in addition to long 3'-overhangs
of the G-rich strand, long 5'-overhangs of the C-rich strand are
transiently present at the telomere end of chromosomes of proliferating
cells. We also show a direct correlation between 5' C-rich tails and
the proliferative rate of the cell cultures. To account for the above
findings, we propose that the replication fork dissolves when the last
possible RNA primer is assembled on the telomere (Fig.
4). Evidence suggests that a stretch of single-stranded DNA up to the unwinding point is present on the lagging
arm, whereas the helicase occupies a part of the unwound parental
leading strand (31). Consequently, the newly synthesized leading strand
will not be extended up to the unwinding point; the DNA replication
machinery will prime the lagging template strand close to the unwinding
point and ahead of the 3'-end of the nascent leading strand. The
coordinated synthesis of the leading and lagging strands will leave the
parental C-strand uncopied until a new Okazaki fragment is initiated on
the G-rich strand. Presumably, the DNA replication machinery will be
able to complete leading-strand synthesis only if the parental G-rich
strand is primed at the very chromosome end of a blunt-end telomere or
at a region corresponding to the end of the C-rich strand of a telomere with a 3'-overhang. Priming occurring more centromeric will produce a
C-rich 5'-tail of the leading strand telomere. The length of the
single-stranded G-rich overhang might thus represent the distance from
the last priming event during lagging-strand synthesis at the end of
the chromosome.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Telomere replication. Telomeres normally
interconvert between a capped and a temporally uncapped physical state
that is competent for telomerase action (2) and is presumably adopted
for telomere replication. During progression of the replicative fork,
helicase (the only protein of the DNA replication machinery depicted
here) unwinds DNA at the replication fork moving in a 3' 5'
direction along the parental leading strand. Because the helicase
precedes leading-strand synthesis, the lagging-strand primer is
synthesized ahead of the growing point of the nascent leading strand
(31). To coordinate replication of the leading and lagging strands,
because the C-rich strand is shorter than the G-rich strand, RNA
priming can occur, at best, on that part of the G-strand template that
corresponds to the last few nucleotides of the C-rich strand. As the
leading strand approaches the end of the chromosome, the two parental
strands will dissociate, leaving a 3'-overhang on the lagging-strand
telomere and a 5'-overhang on the leading-strand telomere. The 5'-end
of the leading strand template must be eroded by exonuclease activity
to produce a 3'-overhang, the substrate required by the telomerase
(pathway 1). In cases in which the leading-strand template
is not fully replicated, 5'-end erosion by exonuclease occurs before
completion of replication, resulting in enhanced telomere shortening
(pathway 2).
|
|
It has been proposed that the natural chromosome ends are sequestered
by the t-loop structure that is promoted by TRF1 and TRF2 and
requires single-stranded extension of the TTAGGG sequence (14). Then,
the leading strand telomeres have to transit from a 5'-overhang to a
3'-overhang. Recent experiments conducted using a dominant-negative
mutant of TRF2 revealed that a high number of mitotic cells exhibited
end-to-end chromosomal fusion between leading-strand telomeres (32). If
the remodeling and capping process is impaired, a not-yet-eroded C-rich
5'-tail may find itself in the right polarity to pair to a newly
generated 3'-overhang G-rich tail. In this case, the erroneous repair
that leads to covalent end-joining between leading telomeres will be favored.
It has been argued that the nub of the chromosomal-end replication
problem lies in the inability of DNA polymerase to complete synthesis
of the leading strand (23). Conventional DNA polymerases cannot
synthesize the extreme 5'-ends of a blunt end DNA molecule. Even if an
RNA primer was paired with the extreme 3'-end of its DNA template,
removal of this last RNA primer would give rise to a daughter molecule
with a 5'-terminal gap. Lagging-strand synthesis would not necessarily
be a problem as long as the RNA primer is positioned on the
3'-overhang. However, the leading strand will lose its 3'-overhang upon
replication. Consequently, if a cell replicates without telomerase, it
will divide into two daughter cells having lagging telomeres very
similar to the parental chromosomes and with an overhang as long as the
distance from the 3'-overhang to the last Okazaki fragment and a
leading telomere shortened of the 3'-overhang. Human chromosomes have a
single-stranded 3'-overhang of up to 300 nucleotides that roughly
corresponds to the length of the Okazaki fragment of eukaryotic cells.
Our data support a model in which telomere shortening is primarily caused by the inability to position the last RNA primer on the parental
G-rich strand beyond the double-stranded region of the chromosome end.
We propose that this is a consequence of the stall of the replication
fork before reaching the very chromosome end. Thus, the C-rich parental
strand that was already shorter with respect to the parental G-rich
strand would require a further step to be replicated up to the end. A
remodelling process occurring before this step would further increase
telomere shortening (Pathway 2 of Fig. 4).
 |
ACKNOWLEDGEMENTS |
We thank A.V. Furano for helpful comments and
suggestions and M. Foiani for helpful discussion on DNA replication.
 |
FOOTNOTES |
*
This work was supported by Fondatione per
l'Avantamento della Ricerca in Medícina Molecolare Onlus.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.
¶
To whom correspondence should be addressed: Viale Marx, 43, 00137 Roma, Italy. Tel.: 39-0686090328; Fax: 39-0686090332; E-mail: ettore.dambrosio@ims.rm.cnr.it.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M208939200
 |
ABBREVIATIONS |
The abbreviations used are:
T-OLA, telomeric
oligonucleotide ligation assay;
TRF, TTAGGG repeat-binding
factor.
 |
REFERENCES |
1.
|
Blackburn, E. H.
(1991)
Nature
350,
569-573[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Blackburn, E. H.
(2001)
Cell
106,
661-673[Medline]
[Order article via Infotrieve]
|
3.
|
Zakian, V. A.
(1995)
Science
270,
1601-1607[Abstract]
|
4.
|
Greider, C. W.
(1996)
Annu. Rev. Biochem.
65,
337-365[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Watson, J. D.
(1972)
Nat. New Biol.
239,
197-201[Medline]
[Order article via Infotrieve]
|
6.
|
Olovnikov, A. M.
(1973)
J. Theor. Biol.
41,
181-190[Medline]
[Order article via Infotrieve]
|
7.
|
Lundblad, V.,
and Szostak, J. W.
(1989)
Cell
57,
633-643[Medline]
[Order article via Infotrieve]
|
8.
|
Singer, M. S.,
and Gottschling, D. E.
(1994)
Science
266,
404-409[Medline]
[Order article via Infotrieve]
|
9.
|
Greider, C. W.,
and Blackburn, E. H.
(1989)
Nature
337,
331-337[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Morin, G. B.
(1989)
Cell
59,
521-529[Medline]
[Order article via Infotrieve]
|
11.
|
Counter, C. M.,
Avilion, A. A.,
LeFeuvre, C. E.,
Stewart, N. G.,
Greider, C. W.,
Harley, C. B.,
and Bacchetti, S.
(1992)
EMBO J.
11,
1921-1929[Abstract]
|
12.
|
Lingner, J.,
Hughes, T. R.,
Shevchenko, A.,
Mann, M.,
Lundblad, V.,
and Cech, T. R.
(1997)
Science
276,
561-567[Abstract/Free Full Text]
|
13.
|
Stansel, R. M.,
de Lange, T.,
and Griffith, J. D.
(2001)
EMBO J.
20,
5532-5540[Abstract/Free Full Text]
|
14.
|
Griffith, J. D.,
Comeau, L.,
Rosenfield, S.,
Stansel, R. M.,
Bianchi, A.,
Moss, H.,
and de Lange, T.
(1999)
Cell
97,
503-514[Medline]
[Order article via Infotrieve]
|
15.
|
Makarov, V.,
Hirose, Y.,
and Langmore, J. P.
(1997)
Cell
88,
657-666[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Wright, W. E.,
Tesmer, V. M.,
Huffman, K. E.,
Levene, S. D.,
and Shay, J. W.
(1997)
Genes Dev.
11,
2801-2809[Abstract/Free Full Text]
|
17.
|
Huffman, K. E.,
Levene, S. D.,
Tesmer, V. M.,
Shay, J. W.,
and Wright, W. E.
(2000)
J. Biol. Chem.
275,
19719-19722[Abstract/Free Full Text]
|
18.
|
Harley, C. B.,
Futcher, B. A.,
and Greider, C. W.
(1990)
Nature
345,
458-460[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Reveal, P. M.,
Henkels, K. M.,
and Turchi, J. J.
(1997)
J. Biol. Chem.
272,
11678-11681[Abstract/Free Full Text]
|
20.
|
Dionne, I.,
and Wellinger, R. J.
(1998)
Nucleic Acids Res.
26,
5365-5371[Abstract/Free Full Text]
|
21.
|
Fan, X.,
and Price, C. M.
(1997)
Mol. Biol. Cell
8,
2145-2155[Abstract/Free Full Text]
|
22.
|
Jacob, N. K.,
Skopp, R.,
and Price, C. M.
(2001)
EMBO J.
20,
4299-4308[Abstract/Free Full Text]
|
23.
|
Lingner, J.,
Cooper, J. P.,
and Cech, T. R.
(1995)
Science
269,
1533-1534[Medline]
[Order article via Infotrieve]
|
24.
|
Lee, M. S.,
Gallagher, R. C.,
Bradley, J.,
and Blackburn, E. H.
(1993)
Cold Spring Harb. Symp. Quant. Biol.
58,
707-718[Medline]
[Order article via Infotrieve]
|
25.
|
Lingner, J.,
and Cech, T. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10712-10717[Abstract/Free Full Text]
|
26.
|
Wellinger, R. J.,
Wolfe, A. J.,
and Zakian, V. A.
(1993)
Cell
72,
51-60[Medline]
[Order article via Infotrieve]
|
27.
|
Wellinger, R. J.,
Ethier, K.,
Labrecque, P.,
and Zakian, V. A.
(1996)
Cell
85,
423-433[Medline]
[Order article via Infotrieve]
|
28.
|
Riha, K.,
McKnight, T. D.,
Fajkus, J.,
Vyskot, B.,
and Shippen, D. E.
(2000)
Plant J.
23,
633-641[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Cimino-Reale, G.,
Pascale, E.,
Battiloro, E.,
Starace, G.,
Verna, R.,
and D'Ambrosio, E.
(2001)
Nucleic Acids Res.
29,
e35[Abstract/Free Full Text]1-7
|
30.
|
Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, pp. 9.16-9.19, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
31.
|
Gruber, M.,
Wellinger, R. E.,
and Sogo, J. M.
(2000)
Mol. Cell. Biol.
20,
5777-5787[Abstract/Free Full Text]
|
32.
|
Bailey, S. M.,
Cornforth, M. N.,
Kurimasa, A.,
Chen, D. J.,
and Goodwin, E. H.
(2001)
Science
293,
2462-2465[Abstract/Free Full Text]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.