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Role of dolichol in the control of DNA replication in swiss mouse 3T3 cells
Cell Biology International
Reports, Vol. 14, Abstracts Supplement
THE ANTISBMSE STRAND OF SOYBEAN NODULE DRATR OXIDASE cDNA ENCODES
P193
A DNA BINDING PROTEIN
Enrique Preddie* and Johanna E. Bergmann!. *Probe, Dollard des Ormeaux, P.Q., Canada, and !Molecular Biology Lab, Dental School, University of Hamburg, Hamburg, PRG. The antisense strand of soybean nodule urate oxidase cDNA encodes a 90 amino acid protein (drp90). Immunoblot experiments with anti-root and anti-nodule antibodies show the presence of proteins in the t% range of drp90 (10 - 12 kDa) in uninfected root extracts but not in nodule extracts. Computer analysis of the deduced amino acid sequence predicted structures in drp90 with potential for DNA binding. In order to investigate if drp90 interacts with DNA and hence may be involved in transcription regulation in soybean nodules, the region of urate oxidase cDNA encoding drp90 was excised with PstI and SecI and cloned into the expression plasmid pTrc99B. The construct was transfected into E.coli RB791 which was induced with IPTG to overexpress a 12 kDa fusion protein (drp90f). A crude protsin from bacteria expressfraction obtained ing drp90f retarded electrophoretic mobility on lb agarose gels, of a 300 bp fragment from the 3' untranslated end of urate oxidase cDNA. Drp90 has been purified by DNA-affinity chromatography, and footprinting experiments are being carried out with lbcs 3'5'cat and Nodulin-23 cat in order to provide information on the effect of drp90 on the expression of these genes.
HIGH mRNA
P195
LEVEL
OF DNA
POLYMERASE
/-3
IN PACHYTENE SPERMATOCYTES. A. Siedlecki, Radoslawa Nowak. Wawel ska 15. Center-Institute,
Janusz Cancer
02-034
Warsaw, Changes in gene in developing
investigated. that
the
20
times
Poland the
expression rat
Dot amount
p-p01
of
higher
compared to most The increasing
hZe
testes
hybridization in
mRNA is
adult t i 55ues
rat
level
p-p01
of
p-po1 been reveals
at
least
testes as examined. mRNA during
spermatogenesis is associated with the growing number of pachytene spermatocytes DNA synthesis takes where unscheduled intriguing are However, the most place. the changes in the expression pattern of p-p01
The
gene.
reveals 4.0
two
p-p01 In
kb.
spermatogenesis be
dominant.
By
transcript
kb
during
the
trascripts, the early kb the
1.4 kb 5tage
and of
events
can
transcript level
meiotic in
is of
continuously
first dominant
becomes
mitotic
(when the 4.0 contrast,
observed)
hybridization
Northern
1.4
increases
division
and The
pachytene.
overall increase of the level of this transcript is 30-50 When fold. spermatogenesis is at-rested with busulphan (an inhibitor of spermatogonial mitosis), a significant decrease of the
1.4 P
transcript results suggest
kb
These
is
involved
synthesis the
enzyme
exclusively
during activity
in
level is observed. that DNA polymerase gap-filling DNA
recombination is mainly,
regulated
on
mRNA
and
if level.
that
not
1990
127
TIMING OF fra(3)(p14.2) INDUCTION IN LmPDDUYTRCULTURRS Berardino Porfirio, Marco Seri, and Mario De Marchi. Cattedra di Genetica Wedica, Universita' di Siena, Policlinico "Le Scotte" , I-53100 Sierra, Italia. As a general mechanism for the induction of chromosomal fragile sites (FS), Laird et al. (TIQ 3:274, 1987) proposed that FS are regions where DNA replication is late, even delayed into the 02 phase. This hypothesis is here tested using the DNA polymerase-alpha inhibitor aphidicolin (APG), which is known to elicit chromosomal damage only when cells traverse their S phase. PHA-stimulated lymphocytes were treated with 0.2 uM APG either continuously for the last 26, 23, . . . , 3h of culture, or for a 3h pulse, followed by recovery in APC-free mediuw for 23, 20, . . . . Oh. Mean number of breaks per cell (bpc) and expression of fra(3)(p14.2), the most commonFS (FDAJB), were evaluated in fifty metaphaaes Per point. In the continuous schedule, both bpc and FRA3B expression were dramatically dependent on the timing of APG addition: distinct peaks (-3 bpc and 40% FDA38 expression) were observed in the cultures treated for the last 17 - 14h with APG. Afterwards, both paraaeters progressively decreased, down to 0.10 and 1% in cultures treated for the last 3h. To pinpoint the peak of hypersensitivity, APC pulses spanning various phases of the cell cycle were given. The highest levels of bpc and of FFtA3B expression were achieved by pulses extending from 14 was much less to llh prior to harvest. The inhibitor effective when added both nearer and farther the harvesting time. These findings seea to rule out the late replication hypothesis for the induction of common FS, and suggest that the liable period is rather localized in the early-S phase.
P194
ROLE DF DOLICIWL IN THE CWTROL OF DNA REPLICATION IW SWISSMOUSE 3T3 CELLS Luis Jimenez de Asua, Mercedes Goin, Alvaro Estevez, INGEBI, Obligado 2490, 1428 Buenos Aires, Argentina. The oroliferation of animal cells occurs throuah the orderly expression of a program of biochemical events leading up to DNA replication and cell division. Confluent resting Swiss 3T3 cells can be stimmulated by a variety of growth factors, such as epidermal growth factor (EGEl,prostagIandin F2N(PGF&U and others. Mevinolin, a competitive inhibitorofthe HMCoA reductase enzyme,when added to Swiss 3T3 cell blocks the stimulatory effect of EGFor PGF&in the initiation of DNA replication. The inhibition only occurs if mevinolin is added within O-5 hr of stimulation. Mevanolactone, an analogue of mevalonic acid, reversed the inhibition by mevinolin only if added within O-5 hr after addition of EGF or PGF2q. Later additions of mevanolactone has no effect. Dolichol, one of the final metabolic products, also reverses the inhibitory effect of mevinolin if added 0 to 5 hr after EGF-or PGF20(. Since dolichol,its phosphorylation and elongation with sugars is involved in the synthesis of N'glycoproteins, these results suggest that the synthesis of dolichol as well as N'glycoproteins during early lag phase play an important role in the initiation of DNA replication and cell division. A hypothesis will be discussed. This work was supported by the CONICET and by funds generously given by Richard Dreyfus,Basel, Switzerland.
P196
LJA and MG are Principal Investigator fellow of the CONICET, respectively.
and research
Reports, Vol. 14, Abstracts Supplement
THE ANTISBMSE STRAND OF SOYBEAN NODULE DRATR OXIDASE cDNA ENCODES
P193
A DNA BINDING PROTEIN
Enrique Preddie* and Johanna E. Bergmann!. *Probe, Dollard des Ormeaux, P.Q., Canada, and !Molecular Biology Lab, Dental School, University of Hamburg, Hamburg, PRG. The antisense strand of soybean nodule urate oxidase cDNA encodes a 90 amino acid protein (drp90). Immunoblot experiments with anti-root and anti-nodule antibodies show the presence of proteins in the t% range of drp90 (10 - 12 kDa) in uninfected root extracts but not in nodule extracts. Computer analysis of the deduced amino acid sequence predicted structures in drp90 with potential for DNA binding. In order to investigate if drp90 interacts with DNA and hence may be involved in transcription regulation in soybean nodules, the region of urate oxidase cDNA encoding drp90 was excised with PstI and SecI and cloned into the expression plasmid pTrc99B. The construct was transfected into E.coli RB791 which was induced with IPTG to overexpress a 12 kDa fusion protein (drp90f). A crude protsin from bacteria expressfraction obtained ing drp90f retarded electrophoretic mobility on lb agarose gels, of a 300 bp fragment from the 3' untranslated end of urate oxidase cDNA. Drp90 has been purified by DNA-affinity chromatography, and footprinting experiments are being carried out with lbcs 3'5'cat and Nodulin-23 cat in order to provide information on the effect of drp90 on the expression of these genes.
HIGH mRNA
P195
LEVEL
OF DNA
POLYMERASE
/-3
IN PACHYTENE SPERMATOCYTES. A. Siedlecki, Radoslawa Nowak. Wawel ska 15. Center-Institute,
Janusz Cancer
02-034
Warsaw, Changes in gene in developing
investigated. that
the
20
times
Poland the
expression rat
Dot amount
p-p01
of
higher
compared to most The increasing
hZe
testes
hybridization in
mRNA is
adult t i 55ues
rat
level
p-p01
of
p-po1 been reveals
at
least
testes as examined. mRNA during
spermatogenesis is associated with the growing number of pachytene spermatocytes DNA synthesis takes where unscheduled intriguing are However, the most place. the changes in the expression pattern of p-p01
The
gene.
reveals 4.0
two
p-p01 In
kb.
spermatogenesis be
dominant.
By
transcript
kb
during
the
trascripts, the early kb the
1.4 kb 5tage
and of
events
can
transcript level
meiotic in
is of
continuously
first dominant
becomes
mitotic
(when the 4.0 contrast,
observed)
hybridization
Northern
1.4
increases
division
and The
pachytene.
overall increase of the level of this transcript is 30-50 When fold. spermatogenesis is at-rested with busulphan (an inhibitor of spermatogonial mitosis), a significant decrease of the
1.4 P
transcript results suggest
kb
These
is
involved
synthesis the
enzyme
exclusively
during activity
in
level is observed. that DNA polymerase gap-filling DNA
recombination is mainly,
regulated
on
mRNA
and
if level.
that
not
1990
127
TIMING OF fra(3)(p14.2) INDUCTION IN LmPDDUYTRCULTURRS Berardino Porfirio, Marco Seri, and Mario De Marchi. Cattedra di Genetica Wedica, Universita' di Siena, Policlinico "Le Scotte" , I-53100 Sierra, Italia. As a general mechanism for the induction of chromosomal fragile sites (FS), Laird et al. (TIQ 3:274, 1987) proposed that FS are regions where DNA replication is late, even delayed into the 02 phase. This hypothesis is here tested using the DNA polymerase-alpha inhibitor aphidicolin (APG), which is known to elicit chromosomal damage only when cells traverse their S phase. PHA-stimulated lymphocytes were treated with 0.2 uM APG either continuously for the last 26, 23, . . . , 3h of culture, or for a 3h pulse, followed by recovery in APC-free mediuw for 23, 20, . . . . Oh. Mean number of breaks per cell (bpc) and expression of fra(3)(p14.2), the most commonFS (FDAJB), were evaluated in fifty metaphaaes Per point. In the continuous schedule, both bpc and FRA3B expression were dramatically dependent on the timing of APG addition: distinct peaks (-3 bpc and 40% FDA38 expression) were observed in the cultures treated for the last 17 - 14h with APG. Afterwards, both paraaeters progressively decreased, down to 0.10 and 1% in cultures treated for the last 3h. To pinpoint the peak of hypersensitivity, APC pulses spanning various phases of the cell cycle were given. The highest levels of bpc and of FFtA3B expression were achieved by pulses extending from 14 was much less to llh prior to harvest. The inhibitor effective when added both nearer and farther the harvesting time. These findings seea to rule out the late replication hypothesis for the induction of common FS, and suggest that the liable period is rather localized in the early-S phase.
P194
ROLE DF DOLICIWL IN THE CWTROL OF DNA REPLICATION IW SWISSMOUSE 3T3 CELLS Luis Jimenez de Asua, Mercedes Goin, Alvaro Estevez, INGEBI, Obligado 2490, 1428 Buenos Aires, Argentina. The oroliferation of animal cells occurs throuah the orderly expression of a program of biochemical events leading up to DNA replication and cell division. Confluent resting Swiss 3T3 cells can be stimmulated by a variety of growth factors, such as epidermal growth factor (EGEl,prostagIandin F2N(PGF&U and others. Mevinolin, a competitive inhibitorofthe HMCoA reductase enzyme,when added to Swiss 3T3 cell blocks the stimulatory effect of EGFor PGF&in the initiation of DNA replication. The inhibition only occurs if mevinolin is added within O-5 hr of stimulation. Mevanolactone, an analogue of mevalonic acid, reversed the inhibition by mevinolin only if added within O-5 hr after addition of EGF or PGF2q. Later additions of mevanolactone has no effect. Dolichol, one of the final metabolic products, also reverses the inhibitory effect of mevinolin if added 0 to 5 hr after EGF-or PGF20(. Since dolichol,its phosphorylation and elongation with sugars is involved in the synthesis of N'glycoproteins, these results suggest that the synthesis of dolichol as well as N'glycoproteins during early lag phase play an important role in the initiation of DNA replication and cell division. A hypothesis will be discussed. This work was supported by the CONICET and by funds generously given by Richard Dreyfus,Basel, Switzerland.
P196
LJA and MG are Principal Investigator fellow of the CONICET, respectively.
and research