Introduction/background
Saccharomyces cerevisiae is an organism that can exist either as a singled-cell organism or as pseudomycelia. It is used in many scientific studies. It is commonly known as baker’s yeast. Saccharomyces cerevisiae is widely employed in scientific studies mainly due to the following reasons: it has a sequenced genome, it is easy to maintain in the lab, researchers can easily manipulate its genetics, and it grows rapidly. Saccharomyces cerevisiae can live with two genomes (diploid, n=32) or one genome (haploid, n=16). Consequently, the organism can undergo meiotic differentiation. In diploid phase, cells of the saccharomyces cerevisiae are more resistant to hostile environmental conditions than when in the haploid phase. In suitable environmental conditions, sporulation takes place. Consequently, four haploid yeast cells are formed. They include two a and two α.
Meiosis refers to the process of cell differentiation that leads to the production of four daughter cells with half the number of sets of chromosomes as the parent cells. The process mainly takes place in the reproductive organs of organisms that reproduce through sexual means. Meiosis differs from mitosis in certain senses. First, whereas meiosis leads to the formation of haploid cells, mitosis causes the formation of diploid cells. Secondly, meiosis involves exchange of genetic materials between homologous chromosomes during its prophase phase where two homologous chromosomes form bivalent pair. On the other hand, mitosis does not involve any exchange of genetic materials. Due the possibility of exchange of genetic materials between homologous chromosomes, meiosis leads to diversity. On the other hand, mitosis does not encourage diversity given that the process does not involve the exchange of genetic information between homologous chromosomes. Another aspect that distinguishes meiotic replication from mitotic differentiation is the fact that meiotic replication takes place for a significantly longer duration longer than mitotic replication. In addition, unlike mitosis, the initiation of meiosis takes place after the activation of Clb5 and Clb6 (Stuart and Wittenberg, 1998). Meiotic replication also differs from mitotic replication in terms of the mechanisms that control the removal of Cdk inhibitor Sic1. In this case, unlike in mitotic, meiosis requires the action of Ime2 for the removal of Cdk inhibitor Sci1. Ime2 is a kinase specific to meiosis.
Saccharomyces cerevisiae exhibits a process known as sporulation that is responsible for the reproductive activities of the organism. Sporulation involves cellular differentiation initiated in diploid cells. The initiation of cellular differentiation is triggered by nitrogen starvation, the absence of glucose, and the presence of a non-fermentable source of carbon. Therefore, whenever changes in the nitrogen concentration, absence of glucose, or the presence of non-fermentable source of carbon (IV) oxide occur in the external environment of Saccharomyces cerevisiae, the changes are translated into incorporated cellular response. The response controls the shift from mitotic differentiation to meiotic differentiation (Honigberg and Purnapatre, 2003).
Replication of DNA is one of the earliest processes that take place in the prophase phase of meiosis. Another notable activity that takes place during meiosis is formation of double –strand breaks (DSB). Meiotic replication and endonucleolytic activity aid in this process. If S-phase cyclins are absent, meiotic replication does not occur. Besides, generation of DSB independently from initiation of recombination would not take place. Some studies show that the formation of DSB and DNA replication are related due to reconfiguration of chromatin.
Meiotic differentiation takes place alongside a coordinated transcriptional program. Meiotic genes can be classified into various classes based on their timing of expression. The early class of meiotic genes is stimulated by the transcriptional factor Ime2. Therefore, transcriptional factor Ime2 is required during the initiation of meiosis. Proteins that control the early steps of meiosis are encoded by the early class of meiotic genes. Spore morphogenesis and meiotic nuclear divisions require products of the middle class genes. Transcription factor Ndt80 controls the induction of the middle gene expression.
Set1 protein is a complex of eight proteins. Set1 protein is required for efficient catalysis of lysine 4 on histone H3 (H3-K4) methylation. Every H3-H4 can be di-methylated or trimethylated. Specific biological responses may influence the state of methylation. For instance, transcriptional activation influences transcriptional activation. Studies show that deletion of SET1 causes severe defects of sporulation.
Statement of the problem
Further insight into the role of set1 protein in yeast and its effect on meiosis would enable researchers to manipulate reproduction in the organism in order to simplify studies involving yeast. In addition, if scientists further explore how meiosis can be regulated by manipulating step1 protein, there would be a possibility to manipulate the process of meiosis in yeast to improve operations in biotechnology. Furthermore, it would boost advancement in technology in biotechnology. Findings from the study are also important for simplifying efforts in exploring various phenomena in genetics and biotechnology.
Currently, there are few insights into the role of set1protein in yeast on the process of meiosis. Even though many studies have been conducted in order to explore more insight into the entire process of meiosis, there are still many areas to be investigated.
There is a need to conduct research on how step1 protein influences meiosis. A proven-scientific study is important for providing the basis for further improvement of applications of the scientific findings. Through this study, deeper insight into the entire process of meiosis will be obtained. In addition, the study will provide scientifically-proven basis on which various applications concepts of reproduction and genetics will be applied. In addition, this study will give students opportunity to learn other aspects of the research topic that are equally as important as the topic under study.
Research questions
This research seeks to explore the following questions:
- Does the loss of set1affect the onset of meiotic S phase?
- Does the delay in set1∆ meiotic S-phase affect the defect in DNA replication initiation?
- Can overproduction of the set1 SET domain complement delay in meiotic S-phase in set1∆ cells?
- Does the deletion of set1 affect the formation of DSB in meiotic differentiation?
- Does the absence of set1 protein cause various meiotic defects?
Literature review
Many studies have been conducted to explore the entire process of meiosis in saccharomyces. In addition, studies on various aspects of the process are still being conducted in order to find more insight into the phenomenon.
A study carried out by Sollier and colleagues (2004) offers significant insight into the subject. First, the study found that the delay of meiotic S phase is influenced by the loss of set1. In the study, the researchers induced deletion of SET1 gene in the SK1 background. Repeated comparison of the sporulation in set1∆ diploid cells with sporulation in the wild-type diploid cells revealed that the latter was more efficient than the former.
The study also found that defects in the DNA replication initiation are associated with delay in the set1∆ meiotic S-phase. In this case, Clb5myc accumulation in the set∆ cells showed slight defects. However, the study noted that there was no difference in kinase activity between wild type and set1∆ cells.
The study further found that set1 SET domain overproduction can complement the delay in meiotic S. Phase in set1∆ cells. In this case, the study found that HMTase activity is not exclusively necessary for the replication of meiotic DNA. Therefore, the study concludes that activities of set1 in relation to meiotic replication do not depend on the corresponding HMTase activity.
The study also found that DNA damage checkpoint proteins do not influence delay in meiotic S. phase in set1∆ cells. The study further found that meiotic DSB formation is affected by the loss of set1. In addition, the study found that in order for the induction of the expression of the middle meiotic gene to occur, methylation of H3-K4 should take place. Another study by Briggs and colleagues also sought to investigate the role of set1in histone H3 lysine 4 methylation in saccharomyces cerevisiae. Other findings similar to those derived from the study carried out by Sollier and colleagues were derived from the study. For instance, a study by Briggs (2001), found that methylation of H3 lysine 4 mediated by set1 is necessary for the normal growth of cells. Furthermore, it helps in transcriptional silencing.
A study by Soares and colleagues (2014) found that transcription and methylation of H3K4 stabilizes set1. The study further found that levels of set1 protein are associated by methylation of H3K4. A study by Roguev and colleagues also provides more insight into the role of step1 in meiotic replication in saccharomyces cerevisiae. The study sought to explore the components of set1 complex whereby more insight into the operation of set1are given. Roguev and colleagues outline the biochemical characteristics of set1C. The study provides adequate insight that can help in understanding the mechanism by which set1 influences various aspects of meiosis in saccharomyces cerevisiae.
Sims, Robert, Kenichi, and Reinberg also provide some insight into histone lysine methylation. They explain various functions undertaken by various enzymes in meiosis. Findings from their study provide deeper insight into the role of set1 in the regulation of meiotic differentiation.
A study by Bryk and colleagues (2002) sought to explore the evidence that set1 is important for histone H3 methylation and rDNA silencing regulation in saccharomyces cerevisiae. The study found that DNA silencing relies on set1. However, the study found that mitotic stability of Ty1 elements in the rDNA is not regulated by set1. The study also found that SET1 deletion is not associated with the alteration between sir2 or Net1 and rDNA.
Many studies have been conducted in relation to how ste1 influences meiotic replication. In addition, many studies have also been conducted to explore the mechanisms that are involved in various processes of meiosis. However, there is still a need to explore various aspects of meiosis further in saccharomyces cerevisiae since this would enable researchers to carry out more studies with ease.
Methodology
This study will be conducted in a laboratory since it involves the application of laboratory equipment. Besides, students will have to manipulate the environment of the subject of the study. Experimental studies will be conducted in order to investigate how set1 protein influences various aspects of meiosis in saccharomyces cerevisiae. Cell culture and preparation will be conducted. Cultures will be grown in presporulation medium for the preparation of nuclear. Time course experiments will also be conducted whereby aliquots from the cultures will be obtained at certain time intervals. This will be undertaken after the transfer of nuclear to the sporulation media. FACS analysis will also be conducted. In this case, meiotic cells will be fixed with alcohol. The samples will then be rehydrated in PBS and then inoculated for at least two hours. All the centromeres of the yeast will be delineated with DNA probe. In order to probe the yeast telomeres, two plasmids will be used. The plasmids to be used must contain fragments of X and Y element. In order to determine regions of homologous chromosome pairing, cosmid probes will be used. Zeiss Axioskop 1 epifluorescence microscope will be used to evaluate preparations.
Researchers will also delete SET1 gene in the SK1 background. Then, comparison of sporulation in set1∆ diploid cells and wild-type cells will be made. Efforts will also be made to assess whether defects in DNA replication initiation are associated with delay in meiotic S. phase in set1∆ cells. In this case, the participants will induce sporulation in both wild-type and set1∆ cells. Samples will then be collected at different times. The samples will be analyzed for meiotic replication. In addition, sample analysis will be conducted for Clb5-associated Kinase activity and Orc6 phosphorylation. The phosphorylation of Orc6 will be delayed by two hours in set1∆ cells. The defects of initiation of DNA replication will be assessed through observation of Clb5myc accumulation in set1∆. Kinase activity will be measured based on accumulation of Clb5-immunoprecipitates.
In order to determine whether set1 SET overproduction can complement delay in meiotic S-phase in set1∆ cells, mutants will be introduced within the SET domain of set 1. In determining whether meiotic S. phase delay in set1∆ depends on DNA-damage checkpoint proteins, test will be performed on whether Mecl/Rad53 pathway activation causes delay in the replication of meiotic DNA in set1∆ cells. Inactivation of the MEC1gene will be employed in this case.
Significance of the study
This study will provide new insight and scientific evidence that will guide future studies seeking to explore the use of saccharomyces cerevisiae. Saccharomyces cerevisiae is an organism with features that enable it to be effectively used for most studies that aim at exploring various phenomena in genetics. The proposed study seeks to provide more insight into the study of cell division and all the associated processes. Findings from this study will provide guidance on future studies and applications of various concepts of cell differentiation and genetics. In addition, the study will provide more insight into set1complex and other aspects of meiosis. Therefore, findings from this study can help manipulate certain factors in various organisms in order to improve quality of organisms. In other words, this study can lead to findings that can be applied to advance certain applications within the field of biotechnology. The study will also help solve problems in the society that require technological interventions. In addition, findings from the study will ease the efforts needed for research. Set1 protein is also a major factor in meiosis. Studies based on it can lead to knowledge of better methods of manipulating the process of meiosis in order to solve various problems. This study is also important since the need for more knowledge increase daily as more challenges and the needs to improve normal operations increase. This study will provide more knowledge to leaners that can be used to address some of the ever increasing challenges. In a nutshell, this study is important and will not only help address problems of students, but will also address the problem of the society as a whole.
Works cited
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