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Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae.

Jiménez J, Cid VJ, Cenamor R, Yuste M, Molero G, Nombela C, Sánchez M - J. Cell Biol. (1998)

Bottom Line: This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication.Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant.Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia.

ABSTRACT
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

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(a) EcoRI restriction analysis of PCR products from an  inner fragment of the cdc15-lyt1 allele (lanes 1, 2, and 3 represent  three different amplificates) and the CDC15 gene (lane 4), showing the disappearance of an EcoRI cleavage site in the mutant allele. (b) Sequence comparison of the CDC15-encoded peptide in  the area where the lyt1 mutation maps from the mutant, the wild-type, and the putative homologue in the fission yeast S. pombe  (Sp), cdc7. Underline, EcoRI site in the corresponding DNA sequence; boldface; residues affected by the mutation; asterisks,  conserved residues; dots, structurally similar residues in the S.  cerevisiae and S. pombe sequences.
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Figure 1: (a) EcoRI restriction analysis of PCR products from an inner fragment of the cdc15-lyt1 allele (lanes 1, 2, and 3 represent three different amplificates) and the CDC15 gene (lane 4), showing the disappearance of an EcoRI cleavage site in the mutant allele. (b) Sequence comparison of the CDC15-encoded peptide in the area where the lyt1 mutation maps from the mutant, the wild-type, and the putative homologue in the fission yeast S. pombe (Sp), cdc7. Underline, EcoRI site in the corresponding DNA sequence; boldface; residues affected by the mutation; asterisks, conserved residues; dots, structurally similar residues in the S. cerevisiae and S. pombe sequences.

Mentions: Due to the length of the CDC15 ORF we adopted a strategy for the analysis of the cdc15-lyt1 allele in three subfragments of ∼1 kb each, based on individual substitutions in the CDC15 wild-type allele, complementation experiments and sequencing (Materials and Methods). Analysis of the DNA sequences revealed no differences with the wild-type CDC15 sequence published by Schweitzer and Phillipsen (1991) except for a G to A transition at position 1,229. This observation is consistent with the mechanism of action of ethylmethane sulfonate, the mutagen used by Cabib and Durán (1975) to obtain the cdc15-lyt1 mutant. A restriction map of the gene revealed that the substitution was part of an EcoRI cleavage site. Restriction analysis of PCR products cloned in pBluescript SK disclosed that the inserts amplified from the wild-type strain YPH499 had an EcoRI restriction site that was absent in inserts obtained from reactions in which the L2C24d had been used as template (Fig. 1). Moreover, sequencing of the PCR products from YPH499 genomic DNA showed that position 1,229 is occupied by a G in the wild-type allele, as in the original sequence obtained by Schweitzer and Philippsen (1991). We therefore conclude that the cdc15-lyt1 allele displays a unique point mutation that results in a substitution of the glycine at position 410 by glutamate.


Morphogenesis beyond cytokinetic arrest in Saccharomyces cerevisiae.

Jiménez J, Cid VJ, Cenamor R, Yuste M, Molero G, Nombela C, Sánchez M - J. Cell Biol. (1998)

(a) EcoRI restriction analysis of PCR products from an  inner fragment of the cdc15-lyt1 allele (lanes 1, 2, and 3 represent  three different amplificates) and the CDC15 gene (lane 4), showing the disappearance of an EcoRI cleavage site in the mutant allele. (b) Sequence comparison of the CDC15-encoded peptide in  the area where the lyt1 mutation maps from the mutant, the wild-type, and the putative homologue in the fission yeast S. pombe  (Sp), cdc7. Underline, EcoRI site in the corresponding DNA sequence; boldface; residues affected by the mutation; asterisks,  conserved residues; dots, structurally similar residues in the S.  cerevisiae and S. pombe sequences.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132980&req=5

Figure 1: (a) EcoRI restriction analysis of PCR products from an inner fragment of the cdc15-lyt1 allele (lanes 1, 2, and 3 represent three different amplificates) and the CDC15 gene (lane 4), showing the disappearance of an EcoRI cleavage site in the mutant allele. (b) Sequence comparison of the CDC15-encoded peptide in the area where the lyt1 mutation maps from the mutant, the wild-type, and the putative homologue in the fission yeast S. pombe (Sp), cdc7. Underline, EcoRI site in the corresponding DNA sequence; boldface; residues affected by the mutation; asterisks, conserved residues; dots, structurally similar residues in the S. cerevisiae and S. pombe sequences.
Mentions: Due to the length of the CDC15 ORF we adopted a strategy for the analysis of the cdc15-lyt1 allele in three subfragments of ∼1 kb each, based on individual substitutions in the CDC15 wild-type allele, complementation experiments and sequencing (Materials and Methods). Analysis of the DNA sequences revealed no differences with the wild-type CDC15 sequence published by Schweitzer and Phillipsen (1991) except for a G to A transition at position 1,229. This observation is consistent with the mechanism of action of ethylmethane sulfonate, the mutagen used by Cabib and Durán (1975) to obtain the cdc15-lyt1 mutant. A restriction map of the gene revealed that the substitution was part of an EcoRI cleavage site. Restriction analysis of PCR products cloned in pBluescript SK disclosed that the inserts amplified from the wild-type strain YPH499 had an EcoRI restriction site that was absent in inserts obtained from reactions in which the L2C24d had been used as template (Fig. 1). Moreover, sequencing of the PCR products from YPH499 genomic DNA showed that position 1,229 is occupied by a G in the wild-type allele, as in the original sequence obtained by Schweitzer and Philippsen (1991). We therefore conclude that the cdc15-lyt1 allele displays a unique point mutation that results in a substitution of the glycine at position 410 by glutamate.

Bottom Line: This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication.Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant.Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología II, Facultad de Farmacia.

ABSTRACT
The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37 degreesC, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother-daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

Show MeSH
Related in: MedlinePlus