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The function of a spindle checkpoint gene bub-1 in C. elegans development.

Wang X, Liu M, Li W, Suh CD, Zhu Z, Jin Y, Fan Q - PLoS ONE (2009)

Bottom Line: In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages.We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans.Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Peking University, Beijing, China.

ABSTRACT

Background: The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.

Methods and findings: In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.

Conclusions: Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.

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Sequence Comparison of BUB-1 and Rescue of fw8.(A) Alignment of C. elegans BUB-1 (http://www.wormbase.org/db/seq/protein?name=WP3ACE06251class=Protein), S. cerevisiae BUB1 (http://db.yeastgenome.org/cgi-bin/protein/protein?sgdid=S000003 420), and H. sapiens BUB1A (http://www.ensembl.org/Homo_sapiens/protview? peptide =  ENSP00000 302530). The conserved protein kinase domain of C. elegans BUB-1 is 29% identical with S. cerevisiae BUB1, and 31% identical with H. sapiens BUB1A. The protein sequences were obtained from wormbase, Ensembl, and SGD. BLASTS of two sequences were done using NCBI BLASTP. Multiple sequence alignment was done using ClustalW on the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/index.html), and the shade was added by using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The shade shows the conserved protein sequence. The black line indicates the deletion region of tm2815. (B) Punc-119::bub-1 partially rescued the reduced D-type neuron defect of fw8. Y axis shows the D-type neuron numbers. The bars represent standard deviation (the t test compared to control fw8; juIs76: P<0.001).
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pone-0005912-g002: Sequence Comparison of BUB-1 and Rescue of fw8.(A) Alignment of C. elegans BUB-1 (http://www.wormbase.org/db/seq/protein?name=WP3ACE06251class=Protein), S. cerevisiae BUB1 (http://db.yeastgenome.org/cgi-bin/protein/protein?sgdid=S000003 420), and H. sapiens BUB1A (http://www.ensembl.org/Homo_sapiens/protview? peptide =  ENSP00000 302530). The conserved protein kinase domain of C. elegans BUB-1 is 29% identical with S. cerevisiae BUB1, and 31% identical with H. sapiens BUB1A. The protein sequences were obtained from wormbase, Ensembl, and SGD. BLASTS of two sequences were done using NCBI BLASTP. Multiple sequence alignment was done using ClustalW on the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/index.html), and the shade was added by using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The shade shows the conserved protein sequence. The black line indicates the deletion region of tm2815. (B) Punc-119::bub-1 partially rescued the reduced D-type neuron defect of fw8. Y axis shows the D-type neuron numbers. The bars represent standard deviation (the t test compared to control fw8; juIs76: P<0.001).

Mentions: We tested a set of RNAi clones covering the interval, and found that RNAi escapers of bub-1 led to reduced number of D-type neurons as well as Emb (data not shown). We then sequenced fw5 and fw8, and identified nucleotide alterations in the bub-1 gene in both alleles (Figure 2A). In C. elegans, the bub-1 gene encodes a 987aa protein with a conserved kinase domain at its C-terminal (Figure 2A). The mutations in fw5 and fw8 result in stop codon at W848 and W726, respectively, which produce truncated proteins lacking the kinase domain. We also obtained a deletion mutant, tm2815, which had an in-frame deletion of 105 amino acids from E473 to A586 in the middle of the protein, with unaffected kinase domain (Figure 2A). Homozygous tm2815 animals displayed embryonic arrest, larval arrest and sterility. However, the phenotypes observed in the deletion allele were weaker than those of fw5 or fw8. We also generated tm2815/fw5 and tm2815/fw8 animals and found that a larger number of surviving adult stage animals compared to homozygous fw5 or fw8 (Table 1). This result indicates that tm2815 mutant behaves as a partial loss of function mutation, and fw5 and fw8 are more likely to be mutations of bub-1.


The function of a spindle checkpoint gene bub-1 in C. elegans development.

Wang X, Liu M, Li W, Suh CD, Zhu Z, Jin Y, Fan Q - PLoS ONE (2009)

Sequence Comparison of BUB-1 and Rescue of fw8.(A) Alignment of C. elegans BUB-1 (http://www.wormbase.org/db/seq/protein?name=WP3ACE06251class=Protein), S. cerevisiae BUB1 (http://db.yeastgenome.org/cgi-bin/protein/protein?sgdid=S000003 420), and H. sapiens BUB1A (http://www.ensembl.org/Homo_sapiens/protview? peptide =  ENSP00000 302530). The conserved protein kinase domain of C. elegans BUB-1 is 29% identical with S. cerevisiae BUB1, and 31% identical with H. sapiens BUB1A. The protein sequences were obtained from wormbase, Ensembl, and SGD. BLASTS of two sequences were done using NCBI BLASTP. Multiple sequence alignment was done using ClustalW on the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/index.html), and the shade was added by using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The shade shows the conserved protein sequence. The black line indicates the deletion region of tm2815. (B) Punc-119::bub-1 partially rescued the reduced D-type neuron defect of fw8. Y axis shows the D-type neuron numbers. The bars represent standard deviation (the t test compared to control fw8; juIs76: P<0.001).
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getmorefigures.php?uid=PMC2691579&req=5

pone-0005912-g002: Sequence Comparison of BUB-1 and Rescue of fw8.(A) Alignment of C. elegans BUB-1 (http://www.wormbase.org/db/seq/protein?name=WP3ACE06251class=Protein), S. cerevisiae BUB1 (http://db.yeastgenome.org/cgi-bin/protein/protein?sgdid=S000003 420), and H. sapiens BUB1A (http://www.ensembl.org/Homo_sapiens/protview? peptide =  ENSP00000 302530). The conserved protein kinase domain of C. elegans BUB-1 is 29% identical with S. cerevisiae BUB1, and 31% identical with H. sapiens BUB1A. The protein sequences were obtained from wormbase, Ensembl, and SGD. BLASTS of two sequences were done using NCBI BLASTP. Multiple sequence alignment was done using ClustalW on the EMBL-EBI website (http://www.ebi.ac.uk/clustalw/index.html), and the shade was added by using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). The shade shows the conserved protein sequence. The black line indicates the deletion region of tm2815. (B) Punc-119::bub-1 partially rescued the reduced D-type neuron defect of fw8. Y axis shows the D-type neuron numbers. The bars represent standard deviation (the t test compared to control fw8; juIs76: P<0.001).
Mentions: We tested a set of RNAi clones covering the interval, and found that RNAi escapers of bub-1 led to reduced number of D-type neurons as well as Emb (data not shown). We then sequenced fw5 and fw8, and identified nucleotide alterations in the bub-1 gene in both alleles (Figure 2A). In C. elegans, the bub-1 gene encodes a 987aa protein with a conserved kinase domain at its C-terminal (Figure 2A). The mutations in fw5 and fw8 result in stop codon at W848 and W726, respectively, which produce truncated proteins lacking the kinase domain. We also obtained a deletion mutant, tm2815, which had an in-frame deletion of 105 amino acids from E473 to A586 in the middle of the protein, with unaffected kinase domain (Figure 2A). Homozygous tm2815 animals displayed embryonic arrest, larval arrest and sterility. However, the phenotypes observed in the deletion allele were weaker than those of fw5 or fw8. We also generated tm2815/fw5 and tm2815/fw8 animals and found that a larger number of surviving adult stage animals compared to homozygous fw5 or fw8 (Table 1). This result indicates that tm2815 mutant behaves as a partial loss of function mutation, and fw5 and fw8 are more likely to be mutations of bub-1.

Bottom Line: In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages.We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans.Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Peking University, Beijing, China.

ABSTRACT

Background: The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.

Methods and findings: In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.

Conclusions: Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.

Show MeSH
Related in: MedlinePlus