Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.

1976 ◽  
Vol 24 (1) ◽  
pp. 34-39 ◽  
Author(s):  
H G Gratzner ◽  
A Pollack ◽  
D J Ingram ◽  
R C Leif
Keyword(s):  
Deoxyribonucleic Acid ◽  
De Novo ◽  
Single Cells ◽  
Acid Synthesis ◽  
S Phase ◽  
Cho Cell ◽  
Metaphase Chromosomes ◽  
Time Intervals ◽  
Deoxyribonucleic Acid Synthesis ◽  
Sepharose 4B

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid

1981 ◽  
Vol 1 (11) ◽  
pp. 1038-1047
Author(s):  
S Kawasaki ◽  
L Diamond ◽  
R Baserga
Keyword(s):  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Acid Synthesis ◽  
Simian Virus ◽  
S Phase ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Deoxyribonucleic Acid Synthesis ◽  
G1 Cells

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells

1981 ◽  
Vol 1 (10) ◽  
pp. 932-938
Author(s):  
I Krishnan ◽  
C Baglioni
Keyword(s):  
Deoxyribonucleic Acid ◽  
Acid Synthesis ◽  
Polymerase Activity ◽  
S Phase ◽  
Activity Level ◽  
Fresh Medium ◽  
Growth Status ◽  
Enzyme Degradation ◽  
Deoxyribonucleic Acid Synthesis ◽  
In Cells

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

scholarly journals Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid.

1981 ◽  
Vol 1 (11) ◽  
pp. 1038-1047 ◽  
Author(s):  
S Kawasaki ◽  
L Diamond ◽  
R Baserga
Keyword(s):  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Simian Virus 40 ◽  
Acid Synthesis ◽  
Simian Virus ◽  
S Phase ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Deoxyribonucleic Acid Synthesis ◽  
G1 Cells

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

scholarly journals Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.

1981 ◽  
Vol 1 (10) ◽  
pp. 932-938 ◽  
Author(s):  
I Krishnan ◽  
C Baglioni
Keyword(s):  
Deoxyribonucleic Acid ◽  
Acid Synthesis ◽  
Polymerase Activity ◽  
S Phase ◽  
Activity Level ◽  
Fresh Medium ◽  
Growth Status ◽  
Enzyme Degradation ◽  
Deoxyribonucleic Acid Synthesis ◽  
In Cells

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.

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