A sustainable smart production model for partial outsourcing and reworking.

Smart production plays a significant role to maintain good business terms among supply chain players in different situations. Adjustment in production uptime is possible because of the smart production system. The management may need to reduce production uptime to deliver products ontime. But, a decrement in production uptime reduces the projected production quantity. Then, the management uses a limited investment for pursuing possible alternatives to maintain production schedules and the quality of products. This present study develops a mathematical model for a smart production system with partial outsourcing and reworking. The market demand for the product is price dependent. The study aims to maximize the total profit of the production system. Even in a smart production system, defective production rate may be less but unavoidable. Those defective products are repairable. The model is solved by classical optimization. Results show that the application of a variable production rate of the smart production for variable market demand has a higher profit than a traditional production (52.65%) and constant demand (12.45%).

Product outsourcing policy for a sustainable flexible manufacturing system with reworking and green investment.

Production of defective products is a very general phenomenon. But backorder and shortages occur due to this defective product, and it hampers the manufacturer's reputation along with customer satisfaction. That is why, these outsourced products supply, a portion of required products for in-line production. This study develops a flexible production model that reworks repairable defective products and outsources products to prevent backlogging. A percentage of total in-line production is defective products, which is random, and those defective products are repairable. A green investment helps the reworking process, which has a direct impact on the market demand for products. A classical optimization solves the profit maximization model, and a numerical method proves the global optimal solutions. Sensitivity analysis, managerial insights, and discussions provide the highlights and decision-making strategies for the applicability of this model.

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, 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 ~ 105 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.

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements.

An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a 'segrosome' by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of 'chromosome hitchhiking' by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the 'mother bias' experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids-at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.

Conformational flexibility of histone variant CENP-ACse4 is regulated by histone H4: A mechanism to stabilize soluble Cse4.

The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.

An interplay between Shugoshin and Spo13 for centromeric cohesin protection and sister kinetochore mono-orientation during meiosis I in Saccharomyces cerevisiae.

Meiosis is a specialized cell division process by which haploid gametes are produced from a diploid mother cell. Reductional chromosome segregation during meiosis I (MI) is achieved by two unique and conserved events: centromeric cohesin protection (CCP) and sister kinetochore mono-orientation (SKM). In Saccharomyces cerevisiae, a meiosis-specific protein Spo13 plays a role in both these centromere-specific events. Despite genome-wide association of Spo13, we failed to detect its function in global processes such as cohesin loading, cohesion establishment and homologs pairing. While Shugoshin (Sgo1) and protein phosphatase 2A (PP2ARts1) play a central role in CCP, it is not fully understood whether Spo13 functions in the process through a Sgo1- PP2ARts1-dependent or -independent mechanism. To delineate this and to find the relative contribution of each of these proteins in CCP and SKM, we meticulously observed the sister chromatid segregation pattern in the wild type, sgo1Δ, rts1Δ and spo13Δ single mutants and in their respective double mutants. We found that Spo13 protects centromeric cohesin through a Sgo1- PP2ARts1-independent mechanism. To our surprise, we observed a hitherto unknown role of Sgo1 in SKM. Further investigation revealed that Sgo1-mediated recruitment of aurora kinase Ipl1 to the centromere facilitates monopolin loading at the kinetochore during MI. Hence, this study uncovers the role of Sgo1 in SKM and demonstartes how the regulators (Sgo1, PP2ARts1, Spo13) work in a coordinated manner to achieve faithful chromosome segregation during meiosis, the failure of which leads to aneuploidy and birth defects.

The 2 micron plasmid: a selfish genetic element with an optimized survival strategy within Saccharomyces cerevisiae.

Since its discovery in the early 70s, the 2 micron plasmid of Saccharomyces cerevisiae continues to intrigue researchers with its high protein-coding capacity and a selfish nature yet high stability, earning it the title of a 'miniaturized selfish genetic element'. It codes for four proteins (Rep1, Rep2, Raf1, and Flp) vital for its own survival and recruits several host factors (RSC2, Cohesin, Cse4, Kip1, Bik1, Bim1, and microtubules) for its faithful segregation during cell division. The plasmid maintains a high-copy number with the help of Flp-mediated recombination. The plasmids organize in the form of clusters that hitch-hike the host chromosomes presumably with the help of the plasmid-encoded Rep proteins and host factors such as microtubules, Kip1 motor, and microtubule-associated proteins Bik1 and Bim1. Although there is no known yeast cell phenotype associated with the 2 micron plasmid, excessive copies of the plasmid are lethal for the cells, warranting a tight control over the plasmid copy number. This control is achieved through a combination of feedback loops involving the 2 micron encoded proteins. Thus, faithful segregation and a concomitant tightly controlled plasmid copy number ensure an optimized benign parasitism of the 2 micron plasmid within budding yeast.

A comprehensive model to predict mitotic division in budding yeasts.

High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division.

Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae.

Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (p<0.05). All the significant spots were reproducible in all biological replicates and followed the same trend. Identification of the proteins from these spots revealed that nine proteins were common in both 2-DE and 2D-DIGE. These proteins are found to be involved in various cellular processes and pathways such as cytoskeleton function and cytokinesis, carbon, nitrogen, lipid metabolism, general translation and protein folding. Among these, our further study with the cytoskeletal proteins reveals that, compared to mitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. BIOLOGICAL SIGNIFICANCE: Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin cytoskeleton and its regulators accumulated during meiosis by forming stable protein structure even though the corresponding mRNAs are degraded as cells enter into meiosis. This is in accordance with recent studies in higher eukaryotes where actin cytoskeleton is found to play vital role during meiotic chromosome segregation. Information generated by this study is significant to reveal that even though a cell that, unlike mitosis, is metabolically inactive with no isotropic bulging of membranes as buds (in meiosis) can require more actin cytoskeleton presumably to support nuclear movements.

Cohesin: functions beyond sister chromatid cohesion.

Faithful segregation of chromosomes during mitosis and meiosis is the cornerstone process of life. Cohesin, a multi-protein complex conserved from yeast to human, plays a crucial role in this process by keeping the sister chromatids together from S-phase to anaphase onset during mitosis and meiosis. Technological advancements have discovered myriad functions of cohesin beyond its role in sister chromatid cohesion (SCC), such as transcription regulation, DNA repair, chromosome condensation, homolog pairing, monoorientation of sister kinetochore, etc. Here, we have focused on such functions of cohesin that are either independent of or dependent on its canonical role of sister chromatid cohesion. At the end, human diseases associated with malfunctioning of cohesin, albeit with mostly unperturbed sister chromatid cohesion, have been discussed.

Iml3p, a component of the Ctf19 complex of the budding yeast kinetochore is required to maintain kinetochore integrity under conditions of spindle stress.

The Ctf19 multi-protein complex of the central kinetochore in Saccharomyces cerevisiae is required for precise chromosome segregation during mitosis. Of 11 proteins of this complex, at least six are required for cell survival when microtubules are defective. To find individual roles of these proteins in kinetochore stability, double deletion mutants of the corresponding genes were constructed in several combinations. The growth phenotype of all the mutants was in accordance with the current model of hierarchical assembly of kinetochore proteins except one that lacked CHL4 and IML3 genes in a tubulin-defective background (tub1-1 chl4 iml3). tub1-1 chl4 iml3 showed synergistic growth defect, decrease in minichromosome stability compared with its single mutants, and a greater accumulation of cells at the G2/M checkpoint of the cell cycle. Furthermore, in the absence of Iml3p, the two-hybrid interaction between Ctf19p (a member of the Ctf19 complex) and Dam1p (a member of the outer kinetochore DASH complex) was disrupted and the localization of Dam1p at the kinetochore was also compromised. These results indicate a role for Iml3p distinct from Chl4p at the kinetochore. Iml3p may be acting as a link between the central and the outer complexes thus contributing to a functional kinetochore.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus.

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the 'partitioning complex' is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

Cohesin: a guardian of genome integrity.

Ability to reproduce is one of the hallmark features of all life forms by which new organisms are produced from their progenitors. During this process each cell duplicates its genome and passes a copy of its genome to the daughter cells along with the cellular matrix. Unlike bacteria, in eukaryotes there is a definite time gap between when the genome is duplicated and when it is physically separated. Therefore, for precise halving of the duplicated genome into two, it is required that each pair of duplicated chromosomes, termed sister chromatids, should be paired together in a binary fashion from the moment they are generated. This pairing function between the duplicated genome is primarily provided by a multimeric protein complex, called cohesin. Thus, genome integrity largely depends on cohesin as it ensures faithful chromosome segregation by holding the sister chromatids glued together from S phase to anaphase. In this review, we have discussed the life cycle of cohesin during both mitotic and meiotic cell divisions including the structure and architecture of cohesin complex, relevance of cohesin associated proteins, mechanism of cohesin loading onto the chromatin, cohesion establishment and the mechanism of cohesin disassembly during anaphase to separate the sister chromatids. We have also focused on the role of posttranslational modifications in cohesin biology. For better understanding of the complexity of the cohesin regulatory network to the readers, we have presented an interactome profiling of cohesin core subunits in budding yeast during mitosis and meiosis.

Cse4 (CenH3) association with the Saccharomyces cerevisiae plasmid partitioning locus in its native and chromosomally integrated states: implications in centromere evolution.

The histone H3 variant Cse4 specifies centromere identity in Saccharomyces cerevisiae by its incorporation into a special nucleosome positioned at CEN DNA and promotes the assembly of the kinetochore complex, which is required for faithful chromosome segregation. Our previous work showed that Cse4 is also associated with the partitioning locus STB of the 2µm circle--a multicopy plasmid that resides in the yeast nucleus and propagates itself stably. Cse4 is essential for the functional assembly of the plasmid partitioning complex, including the recruitment of the yeast cohesin complex at STB. We have located Cse4 association strictly at the origin-proximal subregion of STB. Three of the five directly repeated tandem copies of a 62-bp consensus sequence element constituting this region are necessary and sufficient for the recruitment of Cse4. The association of Cse4 with STB is dependent on Scm3, the loading factor responsible for the incorporation of Cse4 into the CEN nucleosome. A chromosomally integrated copy of STB confers on the integration site the capacity for Cse4 association as well as cohesin assembly. The localization of Cse4 in chromatin digested by micrococcal nuclease is consistent with the potential assembly of one Cse4-containing nucleosome, but not more than two, at STB. The remarkable ability of STB to acquire a very specialized, and strictly regulated, chromosome segregation factor suggests its plausible evolutionary kinship with CEN.

Centromere identity: a challenge to be faced.

The centromere is a genetic locus, required for faithful chromosome segregation, where spindle fibers attach to the chromosome through kinetochore. Loss of centromere or formation of multiple centromeres on a single chromosome leads to chromosome missegregation or chromosome breakage, respectively, which are detrimental for fitness and survival of a cell. Therefore, understanding the mechanism of centromere locus determination on the chromosome and perpetuation of such a locus in subsequent generation (known as centromere identity) is very fundamental to combat conditions like aneuploidy, spontaneous abortion, developmental defects, cell lethality and cancer. Recent studies have come up with different models to explain centromere identity. However, the exact mechanism still remains elusive. It has been observed that most eukaryotic centromeres are determined epigenetically rather than by a DNA sequence. The epigenetic marks that are instrumental in determining centromere identity are the histone H3 variant, CENP-A and the specialized posttranslational modification of the core histones. Here we will review the recent studies on the factors responsible for generating unique centromeric chromatin and how it perpetuates during cell division giving the present-day models. We will further focus on the probable mechanism of de novo centromere formation with an example of neocentromere. As a matter of similitude, this review will include marking extrachromosomal chromatin to be served as a partitioning locus by deposition of CENP-A homolog in budding yeast.

The centromere-specific histone variant Cse4p (CENP-A) is essential for functional chromatin architecture at the yeast 2-microm circle partitioning locus and promotes equal plasmid segregation.

The centromere protein A homologue Cse4p is required for kinetochore assembly and faithful chromosome segregation in Saccharomyces cerevisiae. It has been regarded as the exquisite hallmark of centromeric chromatin. We demonstrate that Cse4 resides at the partitioning locus STB of the 2-microm plasmid. Cse4p-STB association is absolutely dependent on the plasmid partitioning proteins Rep1p and Rep2p and the integrity of the mitotic spindle. The kinetochore mutation ndc10-1 excludes Cse4p from centromeres without dislodging it from STB. Cse4p-STB association lasts from G1/S through late telophase during the cell cycle. The release of Cse4p from STB chromatin is likely mediated through spindle disassembly. A lack of functional Cse4p disrupts the remodeling of STB chromatin by the RSC2 complex, negates Rep2p binding and cohesin assembly at STB, and causes plasmid missegregation. Poaching of a specific histone variant by the plasmid to mark its partitioning locus with a centromere tag reveals yet another one of the molecular trickeries it performs for achieving chromosome- like fidelity in segregation.

Mechanisms for chromosome and plasmid segregation.

The fundamental problems in duplicating and transmitting genetic information posed by the geometric and topological features of DNA, combined with its large size, are qualitatively similar for prokaryotic and eukaryotic chromosomes. The evolutionary solutions to these problems reveal common themes. However, depending on differences in their organization, ploidy, and copy number, chromosomes and plasmids display distinct segregation strategies as well. In bacteria, chromosome duplication, likely mediated by a stationary replication factory, is accompanied by rapid, directed migration of the daughter duplexes with assistance from DNA-compacting and perhaps translocating proteins. The segregation of unit-copy or low-copy bacterial plasmids is also regulated spatially and temporally by their respective partitioning systems. Eukaryotic chromosomes utilize variations of a basic pairing and unpairing mechanism for faithful segregation during mitosis and meiosis. Rather surprisingly, the yeast plasmid 2-micron circle also resorts to a similar scheme for equal partitioning during mitosis.

The Iml3 protein of the budding yeast is required for the prevention of precocious sister chromatid separation in meiosis I and for sister chromatid disjunction in meiosis II.

The mitotic kinetochore of the budding yeast contains a number of proteins which are required for chromosome transmission but are non-essential for vegetative growth. We show that one such protein, Iml3, is essential for meiosis, in that the absence of this protein results in reduced spore viability, precocious sister chromatid segregation of artificial and natural chromosomes in meiosis I and chromosome non-disjunction in meiosis II.