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Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Su TT, O'Farrell PH - J. Cell Biol. (1998)

Bottom Line: We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes.We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles.These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143-0448, USA.

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

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Induction of DNA replication by  cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin  E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or  from heat-shocked larvae at various times after heat shock (in h, indicated above each  lane). Extracts were separated on denaturing  gels and immunoblotted using a previously  characterized antiserum against cyclin E  (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other  lanes contain extract from one pair of salivary  glands. The positions of molecular mass  markers, in kD, are indicated on the side. (B)  Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were  dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock  as indicated above the lanes (min or h:min).  Incorporated BrdU was detected immunologically and DNA was stained with Hoechst  33258. −hs: no heat shock control.
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Figure 2: Induction of DNA replication by cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or from heat-shocked larvae at various times after heat shock (in h, indicated above each lane). Extracts were separated on denaturing gels and immunoblotted using a previously characterized antiserum against cyclin E (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other lanes contain extract from one pair of salivary glands. The positions of molecular mass markers, in kD, are indicated on the side. (B) Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock as indicated above the lanes (min or h:min). Incorporated BrdU was detected immunologically and DNA was stained with Hoechst 33258. −hs: no heat shock control.

Mentions: Analysis of Drosophila embryos indicate that cyclin E is essential for embryonic endocycles. It is expressed in transient pulses each of which overlaps the beginning of each endocycle S phase, and cyclin E mutant embryos fail to undergo endoreplication (Knoblich et al., 1994; Duronio and O'Farrell, 1995). Moreover, production of Drosophila cyclin E from a heat-inducible transgene can drive endocycling cells of the embryo into a synchronous S phase (Knoblich et al., 1994; Duronio and O'Farrell, 1995). We asked if a similar production of cyclin E can induce synchronous DNA synthesis in endocycling cells of early third-instar larvae. To induce cyclin E, larvae carrying the appropriate transgene were subjected to a 37°C heat pulse for 30 min. Salivary glands were dissected at various times after heat shock to monitor cyclin E induction by immunoblotting (Fig. 2 A), or to detect DNA synthesis by labeling with a nucleotide analogue, BrdU, for 15 min. The labeled glands were then fixed, and incorporated BrdU was detected by immunostaining (Fig. 2 B).


Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Su TT, O'Farrell PH - J. Cell Biol. (1998)

Induction of DNA replication by  cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin  E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or  from heat-shocked larvae at various times after heat shock (in h, indicated above each  lane). Extracts were separated on denaturing  gels and immunoblotted using a previously  characterized antiserum against cyclin E  (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other  lanes contain extract from one pair of salivary  glands. The positions of molecular mass  markers, in kD, are indicated on the side. (B)  Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were  dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock  as indicated above the lanes (min or h:min).  Incorporated BrdU was detected immunologically and DNA was stained with Hoechst  33258. −hs: no heat shock control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140170&req=5

Figure 2: Induction of DNA replication by cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or from heat-shocked larvae at various times after heat shock (in h, indicated above each lane). Extracts were separated on denaturing gels and immunoblotted using a previously characterized antiserum against cyclin E (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other lanes contain extract from one pair of salivary glands. The positions of molecular mass markers, in kD, are indicated on the side. (B) Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock as indicated above the lanes (min or h:min). Incorporated BrdU was detected immunologically and DNA was stained with Hoechst 33258. −hs: no heat shock control.
Mentions: Analysis of Drosophila embryos indicate that cyclin E is essential for embryonic endocycles. It is expressed in transient pulses each of which overlaps the beginning of each endocycle S phase, and cyclin E mutant embryos fail to undergo endoreplication (Knoblich et al., 1994; Duronio and O'Farrell, 1995). Moreover, production of Drosophila cyclin E from a heat-inducible transgene can drive endocycling cells of the embryo into a synchronous S phase (Knoblich et al., 1994; Duronio and O'Farrell, 1995). We asked if a similar production of cyclin E can induce synchronous DNA synthesis in endocycling cells of early third-instar larvae. To induce cyclin E, larvae carrying the appropriate transgene were subjected to a 37°C heat pulse for 30 min. Salivary glands were dissected at various times after heat shock to monitor cyclin E induction by immunoblotting (Fig. 2 A), or to detect DNA synthesis by labeling with a nucleotide analogue, BrdU, for 15 min. The labeled glands were then fixed, and incorporated BrdU was detected by immunostaining (Fig. 2 B).

Bottom Line: We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes.We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles.These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143-0448, USA.

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Show MeSH
Related in: MedlinePlus