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Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)

BACKGROUND: Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes,...

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Autores principales: Bharati, Akhilendra Pratap, Ghosh, Santanu Kumar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806570/
https://www.ncbi.nlm.nih.gov/pubmed/31640543
http://dx.doi.org/10.1186/s12860-019-0231-2
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author Bharati, Akhilendra Pratap
Ghosh, Santanu Kumar
author_facet Bharati, Akhilendra Pratap
Ghosh, Santanu Kumar
author_sort Bharati, Akhilendra Pratap
collection PubMed
description BACKGROUND: Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors. RESULT: We constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 10(5) transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype. CONCLUSION: So far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis.
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spelling pubmed-68065702019-10-28 Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM) Bharati, Akhilendra Pratap Ghosh, Santanu Kumar BMC Mol Cell Biol Methodology Article BACKGROUND: Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors. RESULT: We constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 10(5) transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype. CONCLUSION: So far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis. BioMed Central 2019-10-22 /pmc/articles/PMC6806570/ /pubmed/31640543 http://dx.doi.org/10.1186/s12860-019-0231-2 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Bharati, Akhilendra Pratap
Ghosh, Santanu Kumar
Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title_full Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title_fullStr Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title_full_unstemmed Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title_short Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM)
title_sort construction of strains to identify novel factors for regulation of centromeric cohesion protection (ccp) and sister kinetochore mono-orientation (skm)
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806570/
https://www.ncbi.nlm.nih.gov/pubmed/31640543
http://dx.doi.org/10.1186/s12860-019-0231-2
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