I realized while working on a project on CMT 1a, specifically a project where I have to describe the lives of those who are affected by CMT1A, that the clinical descriptions and the stories that the patients tell each other and others do not quite line up. While the descriptions given by doctors are cold and clinical, as they should be there is so much more than the description of the disease in patients who were affected by this disorder. For example, some of the classic symptoms are muscle loss feet and extremities deformities and loss of sensation. While this is interesting from a clinical point of view, this is made even more interesting hearing the patient's personal account of how this affects them personally and how the severity of different symptoms causes trouble or at times odd moments of hilarity in their daily lives. according to the patients because there are feet deformities, if they walk very long, they are liable to have their feet bleed. While this sounds horrifying to the people who do not have the disease to those who do, it is their daily lives, and since the disease also leads to a loss of sensation, this means that they cannot feel it. Symptoms can provide moments of levity as well. few people have told stories of friends or family trying to shock people using a shocking pen, and it not affect the person who was affected at all. Another thing that surprised me is how much the patients were knowledgeable about the drugs that were in a trial in order to treat this disease and their willingness to go any length to get better. In treating CMT, there is a drug combination treatment that is called PTX3003, which consists of three drugs, and laxative, an anti-addiction medication, and antiseizure medication. through the use of these three drugs, studies have proven that it can alleviate the symptoms of CMT 1A. The medicine is currently in phase 3 trials. However, since there are improvements in phase 2 trials, there are patients who have got their doctors to prescribe the three medication that makes up PXT3003 and take them with some visible improvement. I think that as I learn more about medicine and how it works as well as the process in which science is created, I realize more and more that it is incredibly human and relies on so many soft skills to get anything done. this is endlessly frustrating for someone like me tho is not sociable, but it is also a reminder that scientists may have the image of logic, they are human and will have flaws stemming from that.
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Genomic analysis is the identification, comparison of genetic features and their expression through the use of techniques such as DNA sequencing and bioinformatics. Genomic analysis is generally considered to be divided into two categories; structural genomics which identifies certain genomic structures in the genome and functional genomic analysis which analyses the expression of genes and their interactions often also called transcriptomes. Genomic analysis was made available through the prevalence and availability of gene sequencing. While human genome project sequenced the entire human genome most of the genomic analysis would not be possible would not be possible without the further accessibility of sequencing to the point where currently it is extremely affordable to have a genome sequenced, and other techniques such as next-gen sequencing and whole-genome sequencing that does not depend on the isolation of cells in order to sequence the genes. Because these data that are generated from sequencing are generally put online in databases, these data are both available and able used to be used for purposes that are not intended by the researchers who have originally made the sequence. The data set is so large that a single sequencing can be the basis of several papers. Because of this, there is a data analysis bottleneck where there is so much data that needs to be analyzed but there is not enough time or computing power to analyze the entire genome for all the information that it can yield. Because there are so many different venues that use bioinformatics, there is a major need for people who are able to analyze the data that is available and easily accessible. Degrees in Bioinformatics is quickly becoming one of the highest-paid and available jobs on the market.
Hox genes are a superfamily of genes that specify an organism’s body plans. Within the protein made by the HOX genes, there is a homeobox domain is a domain that controls anatomical development in eukaryotes. In animals, there are 16 major classes of homeodomains. HOX genes were originally discovered in 1994 in fruit flies. The HOX gene is especially important in the regulation of development. A mutation in the HOX gene can have a crippling effect on the development, often resulting in physical disorganizes or termination of pregnancy. Because HOX genes are most vital to the development of the fetus, the HOX gene is mostly conserved across species. A mutation in HOX gene that gets fixed in a population can result in a completely new plan such as those that are present in snakes. In snakes, they specifically have a mutation in the HOX6 gene which causes the snake to lose the forelimb. Because of this, snakes have a widely different body plan compared to those of its close relatives who have forelimbs. In labs, hox gene regulation and expression can be manipulated to make certain body parts grow where there normally isn’t a certain body part. The most famous example of this is in fruit flies where researchers caused legs to grow where the eyes should be by manipulating a hox gene called antp. Experiments show the importance of HOX gene in maintaining the general structure of the body plan.
Future experiments that could be done would be to: replicate the experiment, fully cross all combination of mutants and genotype the yeast colonies. The first experiment that could be done is to replicate the experiment. By replicating the experiment, the results would be more certain. Another experiment that could be done is to cross mutants with the same mating type. By doing this, the genotype of the mutants can be better determined. For example, if unknown a 1 was crossed with it self, and the resulting diploid colony grew well on YED and MV+adenine plate but poorly on MV plate, it can be determined that, the mutation is still in the adenine pathway. Yet another experiment that can be done to confirm the site of mutation is to genotype the colonies. By extracting the DNA, and sequencing the DNA, the exact nature of the mutation, whether it is a spot mutation or a missense mutation can be determined. By doing these additional experiments both phenotypic and genotypic traits of the mutant strains can be determined.
Other limitation of the experiment includes that there was no replication experiment that confirms the result of the experiment, the cross between the unknown mutants were limited, and that the growth of the colonies were left in the incubator for a long time so that determining growth was difficult. The first limitation that there is no replication. Replication of the experiment is important in determining what is happening is not due to chance or due to contamination. However, due to the limited amount of time, a replication was not performed. Another limitation is the cross between the unknown mutants were limited. While unknown alpha 1 and unknown alpha 2 was crossed with unknown a1 nd unknown a2, unknown alpha 1 and unknown a1 did not have the same genotype, as did unknown alpha2 and unknown a 2 because of this, unknown alpha 1 was not able to be crossed with unknown alpha 2 and unknown a2 is not able to be crossed with unknown a2. This limits the amount of analysis that can be done as it may be possible that both as have the mutation at the same gene but it was never shown because those two were never crossed. Another limitation is in determining the growth in MV and MV+adenine plates. Because the plates were left in the incubator for 3 days rather than 1 day like other plates, the growth was hard to determine because there were colonies that had noticeably less growth compared to others. Because those had less growth, it was hard to determine whether those counted as part of the colonies that grew well,especially the lack of growth was so noticeable compared to those that grew well.
Future experiments that could be done would be to replicate the experiment, fully cross all combination of mutants and genotype the yeast colonies. The first experiment that could be done is to replicate the experiment. By replicating the experiment, the certainty in the results can be increased, and the possibility that the results were skewed due to contamination or other factors would be limited. Another experiment that could be done is to cross the as and alphas with each other and themselves. By doing this, the genotype of the mutants can be better determined. For example, if unknown a 1 was crossed with unknown a1, and the resulting diploid colony grew well on YED and MV+adenine plate but poorly on MV plate, it can be determined that even if unknown a 1 does not have a mutation at ade1 or ade2, the mutation is still in the adenine pathway. Yet another experiment that can be done to confirm the site of mutation is to genotype the colonies. By extracting the DNA, running PCR and running the DNA on a gel, a more accurate result can be obtained. In addition, using the purified DNA, the DNA sequence can be determined, and the exact nature of the mutation, whether it is a spot mutation or a missense mutation can be determined.
Unknown 1a and unknown 2a both does not seem to have a mutation at ade1 or ade2 since both of the crosses yielded white diploid colonies that grew well on MV plate and MV+adenine plate. Because of this, it is most reasonable to say that the unknown a mutants were complemented at every mutation and as a result, the resulting diploid cell behaves exactly as a wildtype mutation would. Because of this it can be reasoned that the red pigment that is present in both unknown a and unknown 2a is due to some other pigment intermediate that happens to look similar to that of AIR rather than there being a mutation in either ade1 or 2 which would result in the over accumulation of AIR. This would make sense since the cross with the known mutants would cause the resulting diploid colonies to complement, and the trait would not be able to be observed in MV and MV+adenine plates.
In the cross between the four unknown mutations, it is clear that the four mutations does not have mutations in the same gene since the diploid colonies that are the result of the cross behaves in a similar way as a wild type colony in both the YED, MV and the MV+adenine plate. However, there is no guarantee that the two mutant haploid colonies does not have the same mutation and the mutation is in a gene that is not vital in growing on YED, MV and MV+adenine plate and just could not be seen. In addition, the possibility that the two unknown alphas or two as may have the same mutation was not tested.
While there are many limitations to the experiment that was done, the fact that both positive control and negative control had result that was similar to previous experiments implies that the properties of YED and MV plate and the identity of the known mutants could be sure if the known mutants were different from what was previously used, the result of the cross between known mutants would have been different from previous experiments. In the YED plate especially, the result indicates that there are no excess adenine present that may skew with the result of the experiment since the cross between ade2 alpha and ade2a indicates that if a colony is white the reason is not at the fault of the plates but with the mutant colonies themselves. In the MV plate, the lack of known haploid cells and the ade2alpha ade2a crossing indicates under the condition of the plate, a haploid colonies and colonies that cannot produce adenine will not grow well, and if there was a mistake in the plate then the control would not turn out similar to previous experiments. However, since MV+adenine plate was not used last time, the cross of known haploid mutant cells were not useful in that plate, which is one of the limitations of the experiment.
In this experiment, the goal was to figure out the genotype of the unknown haploid mutants through crossing the haploid cells with known and other unknown haploid cells. In doing this, if the resulting diploid colony failed to complement, that would indicate that the two haploid cells had the same mutation. In addition, if the resulting diploid cells had a color that would highly indicate that the place where the mutation is is an enzyme that digest a red intermediate, such as AIR. By using the coloring of the crosses and different properties of the colony that emerges when grown on different plates, a genotype of the haploid mutants can be guessed.
Looking at the data, it is highly likely that unknown 1 mutant alpha have a mutation at ade2. This is indicated by the diploid cross between unknown alpha 1 and ade 2a. The resulting diploid colony is very similar in property that of ade 2 alpha and ade2a. Because of this, it can be assumed there is a ade2 mutation in unknown alpha 1. The mutation at ade2 would also explain the unknown alpha 1 an ade2a’s diploid colony’s behavior on the MV and MV+adenine plates. In the MV plate the colony was not able to grow since adenine is crucial to the viability of the colony. However it was able to grow on the MV+adenine plate because the only mutation that the cross had was at that specific gene, and as soon as the product of the pathway was supplied to the colony it was able to grow.
In addition to unknown alpha 1, unknown alpha 2 also seems to have a mutation in ade2. This is due to the visible color difference in the cross between unknown alpha 2 and ade2a. However, there is also a difference between this diploid cross and cross between ade2 alpha and ade2a. This is perhaps due to other mutations that unknown alpha 2 have acquired through the UV treatment. If for example another enzyme developed a gain of function mutation that digested AIR, that would explain the pinkish color of the diploid colony, and if the pathway crosses with the adenine pathway again, it would explain why the colony grew well on the MV plate.
In the cross between the unknown mutants, there was not a specific expectation as to what the result of the cross would be. However, I expected that there would be at least one cross that would result in a mutant diploid colony, assuming that the reason for the red color resulted from the intermediate AIR. However, against expectations, none of the diploid crosses between the unknowns resulted in a visible mutant cell in the YED plate, as shown in figure 1 and table 1. In the replica to MV plate, all of the white diploid colonies were expected to grow well on the MV plate, and this was shown in the MV plate as shown in figure 2 and table 2. In the replica of MV+adenine plate it was expected that all of the diploid colonies would grow well and the result would be expected as shown in figure 3, table 3.
In the crosses between the known mutants, the expectation of the mutants and their crosses was that ade1alpha, ade2alpha, ade1a, ade1ax ade1alpha, ade1a xade2alpha,and ade2a x ade1alpha would be white or cream colored on the YED plate while ade2a and ade2alpha x ade2a would be red. The result of the YED plate was as expected as shown in figure 1, table 1. In the MV plate, the expectation from previous experiment was that all of the known haploid mutants and ade1a x ade 1alpha and ade2a and ade2 alpha would not grow well while ade1ax ade 2 alpha ande ade2a and ade1alpha would grow well, and the result was as expected as shown in figure 2 and table 2.
In general, most of the results from the cross between the different unknown mutants were unexpected, with very few colonies behaving as predicted. One major difference between the expectation and the result is in the MV plate where there was more colonies growing than I expected. However, all of the control crosses behaved exactly the same as in previous experiments.
In the cross between the known mutants and unknown 1a, the expectation was that since the unknown 1a had a red color that is similar to that of ade2a, in the cross between unknown 1a and ade2alpha the cross would result in a red colony of diploid colony, and the cross with ade1alpha would result in a white diploid colony. While the cross with ade1 alpha was white as expected, the cross with ade2alpha was also white which was unexpected (figure 1, table1). In the replica of the colonies in the MV plate, the expectation was that the cross between unknown 1a and ade2alpha would not be able to grow well on the MV plate while the cross between unknown 1a and ade1alpha would be able to grow well on the MV plate. While unknown 1a did not grow well on the MV plate, both of the diploid colonies resulting from unknown 1a grew well on the MV plate as shown in figure 2 and table 2. In the MV+adenine plate replica, the expectation was that both of the diploid colonies would be able to grow on the MV+adenine plate. The result was as expected with both of the diploid crosses from unknown 1a and known mutants growing well on the MV+adenine plate as shown in figure 3, table 3.
In the cross between the known mutants and unknown 2a, the expectation for the cross on the YED plate was that since the unknown a is a pink haploid colony, the cross with ade1 would create a visibly pink diploid colony whereas in the cross with ade2, there would be complementation and result in an white colony. However, neither of the crosses resulted in a visibly mutant colony as shown in figure 1 table 1. When the colonies were transferred to the MV plate, the expectation that the diploid cross with ade1a would not be able to grow well. However, both of the diploid crosses grew well on the MV plate, as shown in figure 2 and table2. In the MV+adenine plate, the expectation is that both of the diploid crosses would grow well. The result was as expected, as both of the colonies grew well on the MV+adenine plates, as seen in figure 3 and table 3.
In the cross between unknown 1alpha and the known mutant ade1a and ade2a, the expectation was that barring mutation outside of the adenine pathway, the only reason why the unknown alpha1 would be red was because there is either a mutation in ade1, which would cause the cell to be pink, or ade2 which would cause the colony to be a deep red shade. Since unknown alpha 1 had a pink shade, the expectation was that it would have a mutation in ade1, and therefore in the cross between ade 1a resulted in a diploid colony that was pink, while complementation happens with ade2a and resulted in a white diploid colony. This did not happen. Instead the cross with ade1a resulted in a white colony while a cross with ade2a resulted in a red colony, as seen in figure 1 and indicated in table 1. When the cross with the known mutant was transferred to the MV plate, the expectation was that the cross between unknown alpha 1 and ade2 would grow well and the cross with ade1 would grow poorly since the cross with ade 1 lacks complementation. Again the result did not support the expectation. The cross with ade1 grew well on the MV plate while the cross with ade2 grew poorly as shown in figure 2 and table 2. The expectation for the crosses of unknown alpha 1 was that both of the diploid colony would grow well. However, all three of the colonies grew well on the MV+adenine plates, as shown in figure 3 and table 3.
In the crosses between the known mutants and unknown alpha 2, the expectation for the crosses on the YED plate was that since unknown alpha 2 was pink, similar to unknown alpha 1, the cross with ade1 would produce a pink diploid colony where cross with ade2 would result in complementation and as a result, have a white diploid colony. The result went against the expectations. The cross between unknown alpha 2 and ade 1a resulted in an white colony whereas the cross with ade2a resulted in a pink diploid colony as shown in figure 1 and table1. When the cross was transferred to an MV plate, the expectation was that the cross with ade1 and unknown alpha 2 would grow poorly while the diploid cross with ade 2 would grow well. The results partially reflected this as both of the diploid cross with both ade1 and ade2 grew well, as shown in figure 2 and table 2. In the MV+adenine plate, the expectation was that both of the resulting diploid crosses would be a small white colony. The resulting two diploid cells were able to grow on the MV+adenine plate which was expected (figure 3, table 3).