In this lab trimyristin was isolated from the organic compound nutmeg in the presence of tert-methyl butyl ether. The crude trimyristin product was then recrystallized in the presence of acetone and hydrolyzed in the presence of sodium hydroxide and 95% ethanol and then acidified in the presence of hydrochloric acid. Recrystallized trimyristin was recrystallized a second time in the presence of acetone. The recrystallized product was determined to be trimyristin using melting point analysis with a yield of 23.7% after the first recrystallization and 6.0% after the second recrystallization based on the original amount of nutmeg used in the reaction.
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To a round-bottom flask was added nutmeg (1.00g) and tert-butyl methyl ether (3mL). Heating at boiling point was performed to the solution for 10 minutes. Micro-scale filtration was performed on the solution. Crude product was recrystallized from acetone (2mL) and cooled. To a round-bottom flask was added recrystallized trimyristin (60mg), 6M NaOH (2mL) and 95% ethanol (2mL). The solution was refluxed for 45 minutes. The solution was allowed to cool and poured in a beaker containing water (8mL). To the solution was added HCl (2mL) dropwise and stirred while cooled. Remaining recrystallized trimyristin was recrystallized from acetone (2mL). Melting point of product and both recrystallized trimyristin was determined.
In contrast, experiments have shown that a reduction in telomerase activity and the shorting of telomeres has been associated with aging. This highlights the importance of the maintenance of telomere length and protection in control of the cell cycle life span. Further research on the mechanism of telomere maintenance and shortening could provide important information regarding the development of cancer treatment, anti-aging strategies, and hereditary disease function. As the average life span of humans rapidly increases, research in this field will become even more important.
More insight into the mechanism behind telomere maintenance and replication shed light on its role in cancer as well as aging. It was discovered that in 80-90% of cancers, an increase in telomerase activity can be shown. This indicates the role telomere shortening may play in preventing unwanted mutations and cancer. Once cells divide a certain number of times in healthy cells, unprotected and shortened telomeres trigger senescence and apoptosis. With overactive telomerase, this shortening is prevented and cells would be able to proliferate uncontrollable and an unlimited number of times. This mechanism of induced senescence and apoptosis in cells with improper telomere function appears to be regulated by the tumor suppressor gene p53, which acts by binding damaged DNA and promotes p21 activity, a CDK inhibitor which induces cell arrest pathways. Since p53 function is inhibited in 70-80% of cancers, this provides a major hurdle for biologists moving forward in targeting drug therapies towards cancer cells with impaired telomeres.
The “end-replication problem” is one that puzzled many molecular biologists and biochemists for decades. Since a free 3’ OH group is needed for DNA replication and a new strand of DNA to be synthesized, this creates a problem for the ends of linear DNA molecules, such as those we see in eukaryotic cells. Investigation into this mystery began when Blackburn and Szostak sequenced the ends of linear DNA molecules from Tetrahymena and were able to ligate to the ends of yeast cells DNA. These experiments showed evolutionarily conserved properties of Tetrahymena rDNA end sequences with those similar sequences in yeast cells that led to functional telomeres. They were also able to identify repeated C1-3A sequences in telomeres of both organisms. As a graduate student in Blackburn’s laboratory, Greider was able to identify G-rich rDNA molecules in Tetrahymena that contained the necessary 3-OH group for DNA replication that was complementary to the repeats seen in the telomere sequences. The addition of the G-rich oligonucleotide were also shown in vitro to enzymatically be added to yeast telomeric DNA. At this point, the scientists were able to establish a mechanism for telomere synthesis and maintenance involving the enzyme telomerase as a unique reverse transcriptase with an RNA template and protein components, the mystery had been solved.
In 2009 the Nobel Prize in Physiology or Medicine was awarded to three scientists for their discovery and research in telomeres and the enzyme telomerase. Since Watson and Crick published the structure of our DNA and Meselson and Stahl discovered the mechanism by which our DNA is replicated, a mechanism for how the ends of chromosomes can be replicated and maintained. Through years of work, Elizabeth Blackburn, Jack Szostak and Carol Greider were able to determine the mechanism for the replication of telomere DNA, its conserved nature evolutionarily, and key enzymes involved in the process such as telomerase. There findings could have great potential medical application in such fields as cancer and aging.
In 2009 the Nobel Prize in Physiology or Medicine was awarded to three scientists for their discovery and research in telomeres and the enzyme telomerase. Since Watson and Crick published the structure of our DNA and Meselson and Stahl discovered the mechanism by which our DNA is replicated, a mechanism for how the ends of chromosomes can be replicated and maintained.
In his Nobel lecture, Hartwell provides a well-rounded overview of the history and future of cell cycle regulation research. He also discusses the future of research as it pertains to solving human disease, taking a closer look at gene interactions and how they pertain to phenotypic variation. For Hartwell, yeast as a model organism for the study of not only cell cycle regulation, but a host of different topics in molecular biology can provide insight in the future into human disease. Cell cycle regulation is a complex and important mechanism that is highly conserved throughout eukaryotic lineages and further research and understanding are extremely important for not only molecular biologists, but medical practitioners and evolutionary biologists as well.
Another pivotal part of Hartwell’s lecture is his discussion of effects of mis-regulation of the cell cycle and its consequences regarding genomic stability and fidelity. The hallmark of cancer cells is uncontrolled cell growth and proliferation, but they also divide with far worse fidelity, or accuracy than normal cells. This loss of fidelity leads them to mutate and evolve quicker and makes cancer even harder to treat. Hartwell and Ted Weinert studied genes that could have a role in DNA damage checkpoints of the cell cycle and discovered the RAD9 gene, which when mutated resulted in a 20 fold increase in the rate of chromosome loss. The controlling role of CDK in the cell cycle of frogs and yeast were discovered, and along with their homology, the mechanism by which CDK activity is regulated which is the, “series of regulatory signaling pathways, checkpoints, that keep the cell informed of each event’s progress.”
Hartwell’s main discovery was that of CDC genes and the role they play in such cell cycle events in yeast as budding, DNA synthesis, nuclear division cytokinesis and cell division. Hartwell conducted various experiments arresting yeast cells at different points in the cell cycle, as well as comparing the phenotypic defects of cells mutant for the CDC28 gene and others with wild-type yeast cells. He found that the CDC28 gene was required for the commencement of two pathways. The first involved in budding, nuclear migration, cytokinesis and cell division and the other involving DNA replication, nuclear division and joined the first pathway prior to cytokinesis and cell division. Although not referred to in the paper, I interpreted this as concluding evidence of the CDC28 gene being involved in both asexual and sexual reproduction pathways of yeast. Hartwell did however talk about research done by Duntze and Maney regarding the connection between the secretion of pheromones by M ata cells and control of the cell cycle and specifically inhibition of DNA synthesis. Hartwell states that following his discovery of the CDC28 gene, the work Paul Nurse did regarding cyclin dependent kinases unified the cell cycle field. Nurse worked on the CDC2 gene, which he determined its expression to be the rate-limited step of mitosis in S. pombe.