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Securin is not required for chromosomal stability in human cells.

Pfleghaar K, Heubes S, Cox J, Stemmann O, Speicher MR - PLoS Biol. (2005)

Bottom Line: Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect.This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1.We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Technical University Munich, Munich, Germany.

ABSTRACT
Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(-/-) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

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Verification that the Chromosomally Stable Cells Indeed Lack Part of hSecurin by Analyses of Genomic DNA from Parental HCT116 Cells (+/+) and Chromosomally Stable hSecurin−/− Cells (−/−)(A) Transcript structure of the hSecurin gene with its six exons. The lengths of introns and exons are drawn to scale based on the NCBI 35 assembly of the human genome (http://www.ensembl.org). Exons 2 and 3, with the locations of the respective primer pairs, are depicted enlarged.(B) As a control, PCR analysis was done with primers located in exons 8 and 9 of the p53 gene and resulted in the expected amplification product for both cell lines (lanes 2 and 3). In contrast, PCR with primers PTTG-R6 and PTTG-R1, located in the second and third exon of the hSecurin gene (arrows above exons 2 and 3 in [A]), yielded an amplification product only for the parental HCT116 cells (+/+; lane 5) and not for the chromosomally stable hSecurin−/− cells (−/−; lane 6). Lane 1 shows the 100-bp ladder as a size marker, and lanes 4 and 7 are negative controls for the respective primer pairs.(C) PCR analyses with primers SecP1l, located in exon 2, and SecP2r, located in intron 3–4 (arrows below exons 2 and 3 in [A]), resulted in amplification products with different sizes (lanes 2 and 3), reflecting the deletion of exon 3. Lane 1 shows the 100-bp ladder; lane 4 is the negative control.
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pbio-0030416-g003: Verification that the Chromosomally Stable Cells Indeed Lack Part of hSecurin by Analyses of Genomic DNA from Parental HCT116 Cells (+/+) and Chromosomally Stable hSecurin−/− Cells (−/−)(A) Transcript structure of the hSecurin gene with its six exons. The lengths of introns and exons are drawn to scale based on the NCBI 35 assembly of the human genome (http://www.ensembl.org). Exons 2 and 3, with the locations of the respective primer pairs, are depicted enlarged.(B) As a control, PCR analysis was done with primers located in exons 8 and 9 of the p53 gene and resulted in the expected amplification product for both cell lines (lanes 2 and 3). In contrast, PCR with primers PTTG-R6 and PTTG-R1, located in the second and third exon of the hSecurin gene (arrows above exons 2 and 3 in [A]), yielded an amplification product only for the parental HCT116 cells (+/+; lane 5) and not for the chromosomally stable hSecurin−/− cells (−/−; lane 6). Lane 1 shows the 100-bp ladder as a size marker, and lanes 4 and 7 are negative controls for the respective primer pairs.(C) PCR analyses with primers SecP1l, located in exon 2, and SecP2r, located in intron 3–4 (arrows below exons 2 and 3 in [A]), resulted in amplification products with different sizes (lanes 2 and 3), reflecting the deletion of exon 3. Lane 1 shows the 100-bp ladder; lane 4 is the negative control.

Mentions: In the next step we confirmed that chromosomally stable hSecurin−/− cells were indeed hSecurin deficient. We used PCR primer pairs spanning the second and third exons of the securin gene, as described previously [20] (Figure 3A and 3B). In addition, we designed a primer set flanking exon 3 to specifically demonstrate that the hSecurin−/− cells lack exon 3 of the securin gene (Figure 3A and 3C). Genomic PCR analyses with these primer sets using DNA extracted from chromosomally stable hSecurin−/− cells demonstrated that the cells had indeed a homozygous deletion of the exon 3 region of the hSecurin gene. Primers for exons 8 and 9 of p53 were used as an additional control (Figure 3B).


Securin is not required for chromosomal stability in human cells.

Pfleghaar K, Heubes S, Cox J, Stemmann O, Speicher MR - PLoS Biol. (2005)

Verification that the Chromosomally Stable Cells Indeed Lack Part of hSecurin by Analyses of Genomic DNA from Parental HCT116 Cells (+/+) and Chromosomally Stable hSecurin−/− Cells (−/−)(A) Transcript structure of the hSecurin gene with its six exons. The lengths of introns and exons are drawn to scale based on the NCBI 35 assembly of the human genome (http://www.ensembl.org). Exons 2 and 3, with the locations of the respective primer pairs, are depicted enlarged.(B) As a control, PCR analysis was done with primers located in exons 8 and 9 of the p53 gene and resulted in the expected amplification product for both cell lines (lanes 2 and 3). In contrast, PCR with primers PTTG-R6 and PTTG-R1, located in the second and third exon of the hSecurin gene (arrows above exons 2 and 3 in [A]), yielded an amplification product only for the parental HCT116 cells (+/+; lane 5) and not for the chromosomally stable hSecurin−/− cells (−/−; lane 6). Lane 1 shows the 100-bp ladder as a size marker, and lanes 4 and 7 are negative controls for the respective primer pairs.(C) PCR analyses with primers SecP1l, located in exon 2, and SecP2r, located in intron 3–4 (arrows below exons 2 and 3 in [A]), resulted in amplification products with different sizes (lanes 2 and 3), reflecting the deletion of exon 3. Lane 1 shows the 100-bp ladder; lane 4 is the negative control.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1287505&req=5

pbio-0030416-g003: Verification that the Chromosomally Stable Cells Indeed Lack Part of hSecurin by Analyses of Genomic DNA from Parental HCT116 Cells (+/+) and Chromosomally Stable hSecurin−/− Cells (−/−)(A) Transcript structure of the hSecurin gene with its six exons. The lengths of introns and exons are drawn to scale based on the NCBI 35 assembly of the human genome (http://www.ensembl.org). Exons 2 and 3, with the locations of the respective primer pairs, are depicted enlarged.(B) As a control, PCR analysis was done with primers located in exons 8 and 9 of the p53 gene and resulted in the expected amplification product for both cell lines (lanes 2 and 3). In contrast, PCR with primers PTTG-R6 and PTTG-R1, located in the second and third exon of the hSecurin gene (arrows above exons 2 and 3 in [A]), yielded an amplification product only for the parental HCT116 cells (+/+; lane 5) and not for the chromosomally stable hSecurin−/− cells (−/−; lane 6). Lane 1 shows the 100-bp ladder as a size marker, and lanes 4 and 7 are negative controls for the respective primer pairs.(C) PCR analyses with primers SecP1l, located in exon 2, and SecP2r, located in intron 3–4 (arrows below exons 2 and 3 in [A]), resulted in amplification products with different sizes (lanes 2 and 3), reflecting the deletion of exon 3. Lane 1 shows the 100-bp ladder; lane 4 is the negative control.
Mentions: In the next step we confirmed that chromosomally stable hSecurin−/− cells were indeed hSecurin deficient. We used PCR primer pairs spanning the second and third exons of the securin gene, as described previously [20] (Figure 3A and 3B). In addition, we designed a primer set flanking exon 3 to specifically demonstrate that the hSecurin−/− cells lack exon 3 of the securin gene (Figure 3A and 3C). Genomic PCR analyses with these primer sets using DNA extracted from chromosomally stable hSecurin−/− cells demonstrated that the cells had indeed a homozygous deletion of the exon 3 region of the hSecurin gene. Primers for exons 8 and 9 of p53 were used as an additional control (Figure 3B).

Bottom Line: Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect.This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1.We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Technical University Munich, Munich, Germany.

ABSTRACT
Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(-/-) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

Show MeSH
Related in: MedlinePlus