One of the essential function of an organism is to maintain its genome integrity, in order to survive and pass on its genetic materials to the next generation. However, as the carrier of genomic information, DNA is constantly being damaged by exogenous and endogenous factors. Therefore, through evolution, a variety of mechanisms that counter the effect of DNA damage emerged. In this study, we applied different dosages of UV light and Cisplatin, as DNA-damaging agents to SV40 T antigen immortalized MRC 5 Cells, to examine the various cell responses by assaying the relative concentration of key cell cycle regulatory proteins.
In human body, DNA cells carried are constantly under assaults by exogenous and endogenous agents, causing a significant amount of lesions per day (Lindahl et al., 2000). If those lesions are not treated efficiently and the accumulation of lesions continues, mutations and aberrations of genome may lead to the emergence of many forms of physiological disorders, such as tumorigenesis, Parkinson’s disease and Friedrich’s ataxia (Stratton et al.,2009; Mirkin et al., 2007; Yang et al., 2008).
One of the universal exogenous DNA damaging agent is ultraviolet(UV) light. UV-A and UV-B are its residues that are not absorbed by the ozone layer, and these residues may induce approximately 104 lesions to an exposed cell per hour (Doll et al., 1981). UV light is both a mutagen and potent cytotoxic agent ,which can trigger cell apoptosis by either accumulating DNA lesions or trigger CD95/Fas receptor and induce apoptosis directly (Rehemtulla et al., 1997).
Another well-characterized DNA damaging agent is cisplatin. Unlike UV light, It is usually introduced to the human body during chemotherapy. (Prestayko et al., 1979) Researchers had already identified its DNA damage mediated apoptotic signaling pathway, which involves the TAB1 regulation of proteins’ circuitry (Yan et al., 2013).
While DNA is damaged within a cell, there are many possible cellular responses, such as RNA processing, cell arrest and repair. DNA damage repair mechanisms varies according to the type of DNA damage. Apoptosis can be induced by accumulative DNA lesions to ensure the integrity of genome (Stephen et al., 2009) .
Materials and Methods (Instructed By Practical Handout)
SV40 T antigen immortalised MRC5 cell cultures
SV40 T antigen immortalised MRC5 cells were split into eight experimental dishes with the appropriate medium. Those dishes were then incubated (at 5% CO2, 37oC) for two days and ready for cell damage and protein assay.
UV light and Cisplatin were employed as DNA damaging agents. For UV light, we chose 4 levels of dosages:64 J/m2 , 125 J/m2 , 250 J/m2 and 500J/m2; as for cisplatin, we choose to pipette 6.25uM, 12.5uM and 25uM to cells. All cells are damaged for 22 hours.
Cell harvesting and protein assay
After 22h, cells were harvested and prepared for protein assay. Immunohistochemistry and fluorescence microscopy were employed to indicate the level of proliferating cell nuclear antigen (PCNA), and western blotting technique were used to indicate the level of cyclin D1, geminin, tubulin and ?H2AX for eight cell cultures separately.
Hypothesis, Mechanisms, and Expected Results
The hypothesis being tested were: For a cell culture, the overall type and magnitude of DNA damage response depends solely on the level of DNA damage they receive. According to our theory, while cells receive endurable damage, DNA repair mechanism will be favored statistically, which results in cellular arrest and DNA repair. Whereas, when the DNA damage level exceed a threshold value, cells will tend to proceed to apoptotic pathway.
Since we assume there can only be two types of cell fates: cells suffered DNA lesions will either activate the arrest/repair pathway or they will activate the apoptosis pathway. Therefore, by protein analysis on certain key cell cycle regulatory proteins that are involved in those two pathways, we can see the relative level of protein expression and hence deduce the cell responses at different levels of cell damage.
Here we choose to radiate UV light and pipette cisplatin at various dosages to different cell cultures. For UV light we choose 64 J/m2, 125 J/m2 as low dosages and 250 J/m2 and 500J/m2 as high dosages. As for cisplatin, we choose 6.25uM as low dosage, and 12.5uM and 25uM as high dosages.
During this experiment, we examined the level of four proteins via immunoblotting: the first protein indicator we examined was cyclin D1. It is a protein that can be activated in the arrest and repair pathway. Previous investigations also suggest it will prevent low dosage UV-induced apoptosis and promotes adaptive resistance (Ahmed et al., 2008).
The second protein we assayed was geminin. It was initially identified as an inhibitor of DNA replication (McGarry and Kirschner, 1998). Later investigations identified an increase in geminin level are correlated with increased proliferation of tumor cells, suggested that geminin may play a role in tumorigenesis (Wohlschlegel et al., 2002, Montannari et al., 2005). It seems contradictory though, overexpression of geminin in the Drosophila embryos leads to ectopic neural differentiation and cell apoptosis (Quinn et al., 2001). We believed, the functional role of geminin varies according to their presence in the different phases of the cell cycle. Under normal conditions, the majority of geminin will concentrate in G1 phase to regulate cell proliferation efficiently. Whereas, under pathological conditions, geminin will tend to present in S or G2 phase, where it has little influence on the regulation of cell proliferation.(Shreeram et al., 2002, Yoshida et al., 2004).
The third protein we examined was tubulin. It plays a vital role during mitosis, and it is responsible for the production of spindle fibers in the metaphase (Wang et al., 2014). In this case, tubulin is an indicator of cell proliferation and apoptosis levels.
The last protein we examined via western blotting was ?H2AX, which is a sensitive molecular indicator for DNA damage repair. Because, when a double strand break is detected, one of the early cellular response will be the phosphorylation of H2AX, leads to the formation of ?H2AX (Mah et al., 2010) .
We also employed immunofluorescence technique to examine the level of PCNA in cells suffered DNA damages. Previous studies indicated that while UV light is inducing DNA lesions, p21 will be degraded, induce PCNA activation which then leads to DNA repair. (Soria et al., 2006)
According to the functional mechanisms of different proteins, we would expect to see a relatively higher expression level of geminin,?H2AX, cyclin D1 and PCNA while a cell culture is damaged with low dosages of genotoxic agent, and vice versa. Because theoretically, these proteins are involved in cellular arrest and DNA damage repair responses. We would also expect the level of tubulin to decrease gradually while cell damaging level is increasing. Because DNA damaging agents can induce apoptosis and decrease the tendency of cell proliferation.
The actual outcome of this experiment was astonishing. On the one hand, we acquired a relatively consistent green immunofluorescence assay result (Fig 1). The control dish shows a constant level of PCNA expression. When a low dosage of cisplatin was applied to the cell culture, the expression of PCNA tends to increase (Fig 1b), which suggested more cells were undergoing DNA repair. So far our experimental results are consistent with previous researches (Stelter and Ulrich, 2003; Soria et al., 2006). But, when 12.5uM of cisplatin were applied to the cell culture, the level of immunofluorescence drops dramatically. This inconsistency is likely due to human error during cell harvesting and preparation. Because microscopy indicates there were an unusual amount of crystals forming within 12.5uM of cisplatin slide, suggested cells may be left dried for an extended period of time, and proteins might be denatured. Interestingly, we expected cells to switch entirely to apoptotic pathways when we applied the maximum dosages of cisplatin to a cell culture (25 uM). However, we observed a significantly higher level of green immunofluorescence in Fig 1d, suggested its DNA repair pathway are the most active among all cisplatin treatments.
Cell cultures that were treated with UV light had a similar trend in response: when cells were exposed to 63 J/m2 of UV light, the level of PCNA expression are slightly lower than that of the control dish. As we increase the intensity of UV light to 125 J/m2 and 250 J/m2, the level of PCNA decreases accordingly, indicates that most of the cellular responses had switched to the apoptotic pathway. However, the result of the maximum dosages treatment (500 J/m2) were out of our expectation: like the maximum dosages cisplatin treatment, cells which endures maximum dosages of UV light exposure turn out to have the highest level of PCNA expression.
On the other hand, the result from immunoblotting is not consistent enough to be analyzed. By cross-comparison with other groups, we found out that no group observed any band from tubulin analysis, and there are heavy non-specific binding occurs on all cyclin D1 analysis. These outcomes indicated inappropriate secondary antibodies were selected for CD1/tubulin assay. However, as for ?H2AX and geminin assay, we were misguided by the vague molecular marker; hence we cut on the incorrect side, therefore, there are only a few bands on the edge (fig d).
Previous investigations indicated: there are multiple cellular pathways which enables cells to perform DNA damage repair (Teruaki and David, 2013). Such a response is completed by an orchestration of protein activities, including some of the proteins we analyzed during this experiment, such as PCNA. Some previous studies stated that an increasing level of DNA damage leads to a rising PCNA expression (Balajee et al., 2001). These observations partially agrees with our experimental results. However, by applying a relatively higher dosage of damaging agents, we also realized that, after passing through a certain “threshold”, the rise of genotoxicity could results in a gradual decrease in the level of PCNA, which we thought was due to a decrease in the amount of cells undergoes DNA repair and more cells undergoes apoptosis. This observation was also consistent with past findings, which indicates that PCNA activities can be maximally triggered under low levels of UV radiation(Soria et al., 2006).
However, none of any published reports observed sudden increase of PCNA expression when the level of DNA damage was raised from high levels to maximum dosages. There can be three possible explanations for such observations: the first and most likely explanation is: under low dosages, when DNA were repaired the expression of PCNA drop down to base level, whereas under maximum damage, cellular repair pathways are activated throughout the damaging process. The second possibility is: we made errors during experiment and cell cultures were stained differently. Last but not least, there might be a secondary threshold of DNA damage level that leads to acute cell cycle arrest followed by DNA repairing instead of cell apoptosis. Further studies needs to be conducted to testify our expected results on cyclin D1, geminin, tubulin and ?H2AX.
To improve experimental design, we could devise more appropriate UV dosages to maintain the consistency of the experimental outcomes, because the result showed us UV light possess higher genotoxicity compared to cisplatin. We should also consider cell fates other than repair and apoptosis to post a more realistic hypothesis. Finally, a separate experiment to analyze the effect of time length of cell damaging to the cell responses, can be conducted to backup our theory.
As for experimental procedures, besides avoiding possible human errors, our experimental results could be more convincing if we were able to implement following improvements: firstly, we could split cells into at least 16 dishes. This improvement provides larger sample size and testing capacity. Secondly, microscopes with high magnification could be used to perform cell counting to analyze the level of cell apoptosis and cell arrest quantitatively, instead of speculating based on the level of indicators. Last but not least, a quantitative measurement for protein concentrations, such as UV absorption spectrum and iTRAQ technique, could be applied to produce a more reliable result.
Nowadays, we have made much progress toward the understanding of DNA damage responses. However, Some detailed mechanisms, for example, the regulation of protein activities in DDR and the exact magnitude of cell responses toward DNA damage, still remains mysterious to us. Although our experiment failed to unveil this mystery, many published experimental data do support our theories to some extent. Further investigations should be conducted to fully understand the orchestration of DDR mechanisms, and such knowledge will enable us to develop more effective clinical applications to treat DNA damage induced diseases.
Questions and Answers on DNA and MRNA
Q1. What is the full forms of the following:
D.N.A Deoxyribonucleic (The Free Dictionary, 2015)
MRNA Messenger Ribonucleic Acid (The Free Dictionary, 2015)
TRNA Transfer Ribonucleic Acid (The Free Dictionary, 2015)
Q2. Complete the following sentences
The synthesis of mRNA from DNA is known as:- Transcription (The Free Dictionary, 2015)
The synthesis of proteins from RNA is known as:- Protein Synthesis (The Free Dictionary, 2015)
Q3. Explain the process of Protein Synthesis detailing the role of DNA, mRNA, tRNA and ribosomes.
The process where individual cells make protein is called protein synthesis. DNA and all RNA are both involved in this process. RNA are made by enzymes in the cell’s nucleus which start the process of protein synthesis by way of unwinding the wanted section of the DNA. A copy of one side of the DNA is formed by the RNA, and then is sent to all the other areas of the cell to help to bring altogether the different amino acids that make up proteins. As proteins are synthesised by mechanical and chemical processes within the cells, this process is called protein synthesis. Within the nucleus when the strand of RNA has been made, it is called messenger RNA MRNA. Through opening in the nucleus called pores the MRNA exits the nucleus and goes into the larger area of the cell which is called cytoplasm. As soon as the MRNA leaves the nucleus, it is attracted to a structure called ribosome, which are the cells work station or factory for protein synthesis. Within this point only one sub unit of ribosome is present. Another strand of RNA called transfer RNA, TRNA are activated as the MRNA start to attach to the ribosome sub unit. The TRNA stand finds the correct place to bind to the MRNA, as soon as it finds the place; it binds itself to the MRNA, which on one end holding amino acid. As this happens the other sub unit of ribosome come to completely form a complete structure. Another strand of TRNA appears as the ribosome encircles the strand of RNA. This strand is different from the first strand as it is carrying another amino acid; again the TRNA finds the correct place to bind to the MRNA. With the amino acid as soon as the second strand of TRNA is placed, both of the amino acid joins together aided by the ribosome, and adenosine triphosphate (ATP) with its cellular energy. As this pattern continues and repeats itself the chain of amino acid grows longer. As soon as all the amino acids have been placed in the correct sequence, a three-dimensional chain is made. Once this has happened the protein is complete. When the proteins have been made the two sub-units of ribosome part to be joined later. In many ribosomes within the cell the process of protein synthesis take place there (Wisegeek, 2015).
Q4.What is Point Mutation? Evaluate its effects.
Point mutation is where there is a change within a gene, where one base pair in the DNA sequenced is changed or altered.Point mutation is most commonly the result of mistakes made during the DNA replication, however if DNA is exposed to x-rays and or ultraviolet radiations, this can also bring on point mutation.
The two types of mutations are transition mutation and transversion mutation. Transition mutation happens when a pyrimidine base which is thymine (T) or cytosine (C) changes for another pyrimidine base, or it is when a purine base which is adenine (A) or guanine (G) changes for another purine base. Within a double stranded DNA each base is paired with a certain partner on the corresponding strand. A always pairs with T and C always pairs with G. For example a transition mutation is a GC base pair that swaps an AT base pair. However transversion mutation happens when a purine base changes for a pyrimidine base or the other way around i.e. when a TA or CG swaps for AT pair (Britannica, 2015).
Point mutation can be put in three groups, nonsense mutation which is a code for a stop this can shorten the protein. Missense mutation which is a code for a different amino acid and silent mutation which is a code for the same or a different amino acid with no purposeful change in the protein itself. Missense mutation for example causes sickle cell disease, as the beta haemoglobin gene converts a GAG code into a GTG. This codes the amino acid valine instead of glutamic acid (Answers, 2015).
The Effects of Point Mutation
As all the cells within the body contain DNA therefore there are many places where point mutation can happen. However not all mutations are passed on to the offspring, these do not matter for evolution purposes. The mutations that matter for evolution purposes are those that can be passed on to the offspring, these happen in the reproductive cells eggs and sperm this type of mutation is called germ line mutation. There are three effects of germ line mutation:-
No change happens in the phenotype, this is where there is no effect on the organism. This can happen in a part of the DNA strand where there is no function, or it can happen in a protein coding area but does not affect the amino acid sequence of the protein.
Small changes happen in the phenotype, a single mutation can cause some cats ears to curl backwards.
Significant changes happen in phenotype, this is where significant changes happen within the organism for example sickle cell disease. A single mutation can cause negative effects for the organism for example mutation that cause death, this is called lethal.
Mutations are sometimes stereotyped as unimportant or the cause of genetic disease. While many mutations do have a negative effect, other mutations can have a small positive effect. Mutations that control genes can have a huge and sometimes positive effect. As some areas of the DNA control other genes, that decide when and where other genes are turned on, mutation in these parts can considerably change the way an organism is built. This can cause flow of effects in the behaviour of the genes under its control. Organisms have control genes that shape how the body is laid out i.e. the Hox gene is found in humans and animals. This gene sets out where the head goes and where limbs grow. These control gene build the bodies units i.e. limbs, head etc, therefore evolving a huge change in basic body lay out can be likely by a change in the Hox gene and natural selection (Understanding Evolution, 2015).
Q5. What is the probability of a couple having a boy or a girl?
Female X X
XX = Girl
The probability of a couple having a boy is 50%, the probability of a couple having a girl is 50%.
Q6 What chromosomal abnormalities occur in the inheritance of Down’s syndrome
Down syndrome is a result from trisomy 21, this is where the 21st chromosome has three chromosome and not the normal 2 chromosome. Therefore the individual will have 47 chromosomes and not the normal 46 chromosome. Down syndrome is cause by chromosomal abnormalities that happens randomly in the development stage of reproductive cells within a parent. This normally happens in the egg, but can sometimes happen in the sperm. The mistake in cell division results in a reproductive cell with abnormal number of chromosome, trisomy 21. This condition brings on intellectual disability, a characteristic facial appearance and weak muscle tone, all individual that are effected have cognitive delay (Genetics Home Reference, 2012).
Q7. Explain inheritance of a sex-linked condition with a relevant example
Hemophilia A and hemophilia B are both inherited sex linked traits, where they are inherited through the X linked recessive pattern. The genes that cause this condition are in the X chromosome, which is one half of the two sex chromosomes, the other being the Y chromosome. As males only have one X chromosome, one changed copy of the gene in the cell is enough to cause this condition. As females have two X chromosomes a mutation would have to have happened in both of the copies of the genes to cause this condition. This is very rare to happen in females as both copies of genes would have to have the same mutation happened to them. Therefore it is rare for females to have hemophilia. Fathers cannot pass on the inherited X linked condition to their sons as this is one of the characteristics of X linked inheritance. Females who have this X linked recessive condition have one mutated gene are called carriers. Female’s carriers have normally half the amount of coagulation factor which is enough for blood clotting. Some female’s carriers have less than half of the normal amount of coagulation factor, these carriers are at risk for abnormal bleeding (About Education, 2015).
Q8. Explain the inheritance of either cystic fibrosis or phenylketonuria
Cystic fibrosis is a disorder that causes respiratory failure and vitamin deficiencies. Cystic fibrosis effects the secretion of the body i.e. saliva, mucus, sweat and digestive juices. As this secretion should be thin and watery, they are thick and sticky. This causes passageways to be clogged up within the body which causes damage to the pancreas and lungs. Cystic fibrosis is caused by inheriting defected gene from both parents, known as recessive gene. Cystic fibrous cannot be inherited by the off spring, if only one parent has the gene. If the off spring has only one defective gene then they are carriers of cystic fibrosis, this means that they will have the potential to pass on the defective gene to their off spring (eHow, 2015).
The off spring labelled Ff are carriers, they have one defective gene but do not have the disorder themselves. In the diagram above both parents are carriers, they have one in four chances of producing an off spring that has cystic fibrosis (Bitesize, 2014).
In the diagram above one parent is a carrier, while the other is not. They will not produce an off spring with cystic fibrosis, but are able to produce off springs that are carriers (Bitesize, 2014).
Q9 Describe the process of genetic screening explaining the application of DNA recombinant technology in the treatment of a named medical condition.
Genetic testing is a medical test that can indicate any changes in the chromosomes, genes and proteins. The result of genetic testing can help to confirm or reject an alleged genetic condition, disease that the individual may have suspicion of, or help verify their chances of developing and or passing on a genetic disorder to their off spring.
Some of the methods of genetic testing can be of these. Molecular genetic testing, this is where the medics study a single gene or short lengths of DNA to find defectiveness or mutation that may lead to a genetic disorder. Chromosomal genetic testing, this is where a whole chromosome and or long lengths of DNA to see if there are any sizable genetic changes i.e. extra copy of chromosome. Biochemical genetic testing, this is where the activity level or the amount of proteins are studied; any abnormalities that are detected can indicate genetic disorder (Genetic Home Reference, 2015).
DNA recombinant technology, is a technology that produces DNA artificially. The DNA in living organism has been changed today due to this procedure. This procedure involves taking DNA from two difference sources and merging it together for it to become one single molecule. However this only works when the artificially created DNA has been reproduced, this is known as DNA cloning. The two types of cloning that DNA recombinant technology is used for are, therapeutic cloning and reproductive cloning. The reproductive cloning produces an organism with the exact same genetic information from the one that already is living. This type of cloning has been done with Dolly the sheep. Dolly was the first mammal to be reproduced as a genetic copy. Therapeutic cloning, reproduces tissues and or organ and not the whole organism.
Therapeutic cloning has a great deal of benefit, i.e. if an organ has cancer it can be replaced with one made from the individuals own DNA. This reduces any likely hood of organ rejection within the body when a tissue or organ transplant is preformed (Wisegeek, 2015).
Q10 Discuss the moral and ethical issues raised by DNA recombinant technology
Not everyone is in favour of DNA recombinant technology, as they feel that science is playing god by reproducing unnaturally organisms that are not meant to be. And that science is devaluing the uniqueness of life itself. Also that some DNA work involves destroying embryos, this angers some social activist as this death is bought on intently this could be classed as murder. However scientist, argue that the purpose of DNA recombinant technology is to benefit and save human life, and not to destroy it (Wisegeek, 2015).
The free Dictionary 2015, DNA, [online] available at http://acronyms.thefreedictionary.com/DNA, Last Accessed 18/05/15
The free Dictionary 2015, MRNA, [online] available at http://acronyms.thefreedictionary.com/mRNA, Last Accessed 18/05/15
The free Dictionary 2015, TRNA, [online] available at http://acronyms.thefreedictionary.com/tRNA, Last Accessed 18/05/15
WiseGeek 2015, What is Protein Synthesis, [online] available at http://www.wisegeek.org/what-is-protein-synthesis.htm, Last accessed 19/05/15
Encyclopaedia Britannica 2015, Point Mutation, [online] available at http://www.britannica.com/EBchecked/topic/54744/point-mutation, Last accessed 19/05/15
Answers 2015, What is point mutation, [online] available at http://www.answers.com/Q/What_is_point_mutation, Last accessed 20/05/15
Understanding Evolution 2015, The effects of Mutation, [online] available at http://evolution.berkeley.edu/evolibrary/article/mutations_05, Last accessed 20/05/15
Genetics Home Reference 2012, Down Syndrome, [online] available at http://ghr.nlm.nih.gov/condition/down-syndrome, Last accessed 20/05/15
About Education 2015, Sex linked traits, [online] available at http://biology.about.com/od/genetics/ss/sex-linked-traits.htm, Last accessed 23/05/15
eHow 2015, How is Cystic Fibrosis Inherited, [online] available at http://www.ehow.com/how-does_5049940_cystic-fibrosis-inherited.html, Last accessed 23/05/15
Bitesize 2014, Cystic Fibrosis, [online] available at http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa_pre_2011/celldivision/inheritance4.shtml, Last accessed 23/05/15
Genetic Home Reference 2015, What is genetic testing, [online] available at http://ghr.nlm.nih.gov/handbook/testing/genetictesting, Last accessed 23/05/15
WiseGeek 2015, What is Recombinant DNA Technology, [online] available at http://www.wisegeek.org/what-is-recombinant-dna-technology.htm, Last accessed 23/05/15