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Eukaryon, Vol. 9, March 2013, Lake Forest College Senior Thesis Identifying the Telomerase RNA of Aspergillus nidulans: the generation of a DNA construct and refinement of transformation procedures Saajidha Rizvydeen* Department of Biology Lake Forest College Lake Forest, Illinois 60045 Abstract Telomeres are genomic structures found at the ends of linear chromosomes that protect cells by ensuring the complete transmission of genetic information to future generations. As the cell replicates, the telomeres will naturally shorten, allowing them to be used as a biological clock. The enzyme telomerase, a complex molecule made of a protein (TERT) and an RNA component (TR), regulates telomere length. The TR region of the enzyme contains the template region used to synthesize the telomeric repeats, TTAGGG, which are found in most mammals as well as in our model organism of choice — Aspergillus nidulans. In this project, I attempt to knockout a putative TR template. Fusion PCR was used to synthesize a DNA construct that was transformed into fungal cells and integrated into the genome through homologous recombination. I hypothesize that this putative TR template mutation will result in an abnormal phenotype in the fungal cells that arises from the shortening of telomeres due to the dysfunctional telomerase. Introduction There is a diverse array of biological structures found in all living organism that work together to keep the organism alive. The physical structure of these molecules can allude to their functionality in living systems. The existence and evolution of these structures is regulated by natural selection — a process that leads to the increase in frequency of favorable characteristics depending on environmental conditions. In the field of molecular genetics, the understanding of the structure, composition of chromosomes, and the DNA that makes the molecule; helps sheds light on the regulation techniques used by the cell and the structures overall function. Telomeres, the repetitive sequences of DNA found at the ends of linear chromosomes, compose a unique structure that serves as a protective mechanism that ensures the transmission of genetic information to future generations. During DNA replication in cells, enzymes called polymerases are responsible for synthesizing new DNA strands by adding nucleotides that are complementary to the parent strand in a 5’ to 3’ direction. After cell division, daughter cells inherit double stranded DNA that is composed of one newly synthesized strand and one of the original strands. In what is known as the “end replication problem,” these unidirectional DNA polymerases are not capable of synthesizing the ends of the DNA strands to which they initially bind, leading to a decrease in chromosomal length with each cycle of replication (Blackburn et al., 2006). This decrease in length is expected to have a detrimental effect on the cell, as chromosomes consist of genetic material that contains the instructions for cellular development and function. The presence of telomeres, however, prevents the ________________________________________________ *This author wrote the paper as a senior thesis for biology and received distinction under the direction of Dr. Karen Kirk. loss of essential DNA and thus has a protective function to ensure that vital genes existing close to the terminal ends are not lost with each cell division. Telomere Structure and Function The word telomere, coined by geneticist Hermann Muller in 1938, was derived from the Greek root words telos and meros meaning “end” and “part,” respectively. These words describe the location of these structures in all eukaryotic chromosomes (Muller, 1938). Telomeres were first observed in the 1930’s by Muller who studied chromosomal arrangements in Drosophila and Barbara McClintock who conducted similar research in maize (Muller, 1938; McClintock, 1931). Telomeres are comprised of tandem repeats of a specific sequence and vary greatly in length from one organism to another. Mammalian telomeres contain the repeat sequence 5’ –TTAGGG—3’, with the G nucleotide-rich strand extending beyond its complementary strand as a single stranded overhang (deLange, 2005). Although Muller described telomeres as genes in 1938, they were believed to be transcriptionally silent for many decades. Research now shows that telomere DNA transcribes telomere repeat-containing RNA whose function is largely unknown (Azzalin et al., 2007). McClintock and Muller observed that as broken ends of chromosomes underwent fusions, telomeres were left unaltered (Muller, 1938; McClintock, 1931). The protection of the chromosomal end by telomeres is associated with the presence of telomere-specific proteins that distinguish the end from sites of DNA damage known as double stranded breaks. In humans, the proteins that bind to the telomeric repeats are collectively known as the shelterin complex (deLange, 2005). By distinguishing telomeres, the shelterin complex inhibits the initiation of DNA damage responses such as homology-directed repair and non-homologous end joining (deLange, 2005). The proteins of the shelterin complex also shape the telomere ends into a characteristic lasso-like structure known as a t-loop (deLange, 2005). Like the shelterin complex, this structure is conserved in ciliate, plant, and mammalian telomeres and plays a significant function in regulating telomerase activity at the telomeres (deLange, 2010). Telomerase Telomerase is an enzyme that consists of two components: a protein and a RNA component. In somatic cells, telomerase is found in low and almost undetectable levels. However, it is significantly more active in germline, epithelial, and cancer cells (Zhang et al., 2011). The protein component, known as Telomerase Reverse Transcriptase (TERT), is found in eukaryotic cells where it regulates telomere length and synthesizes the addition of complementary nucleotides. Unlike DNA polymerase that uses a DNA strand as the template, telomerase is a reverse transcriptase that contains an intrinsic RNA that aids in the synthesis of telomeric repeats. A specific series of nucleotides within the Telomerase RNA (TR) act as a complementary template that enables the synthesis and elongation of telomeres by single nucleotide additions (Figure 1). Once a repeat is synthesized, telomerase realigns to the end of this newly synthesized sequence and catalyzes the addition of another telomere repeat complementary to the TR template. Eukaryon, Vol. 9, March 2013, Lake Forest College Figure 1. Telomerase-mediated telomere elongation. Telomerase reverse transcriptase (beige), TERT, works in conjunction with the telomerase RNA component (red), TR, to catalyze the addition of nucleotides to the 3’ telomere end of chromosomal DNA (blue). The TR template, shown here as CCCAAU, serves both as a template for complementary nucleotide addition and aids in alignment of telomerase at the telomere end. Telomeres and Cancer Normal telomere shortening, due to the end replication problem, is advantageous as it operates as a cell lifespan clock that is monitored by telomere length checkpoints (Dahlen et al., 1998). Eventually, the telomere will become so short that it can no longer replicate, this stage of cell life is known as the Hayflick Limit. When the Hayflick limit is reached, the cell will end the replication cycle and cell apoptosis will be induced. (Lin & Yan, 2005; Lustig, 1999). However, cells that surpass this regulatory stage and continually proliferate with increasingly shorter telomeres will ultimately reach a “crisis” stage where chromosomal abnormalities, due to loss of essential terminal genes, and high rates of cell death are observed (Lustig, 1999). Most cancerous cells are capable of surpassing the proliferation barrier. Through a series of mechanisms that includes the activation of the enzyme telomerase;the cell’s telomeres are elongated and maintained at long lengths (Lin, 2005). Studies have shown that approximately 85% of all human tumors contain detectable amounts of telomerase activity (Shay & Bacchetti, 1997). Telomerase allows the cell to continually proliferate, accommodating for the decrease in telomere length that arises with normal cell replication. Telomerase RNA Unlike the highly conserved structure of TERT, TR varies greatly both in sequence and in length (Zhang et al., 2011). TR is known to be significant in humans; as its deficiency has been associated with inheritable diseases such as dyskeratosis congenita, aplastic anemia, myelodysplasia, and idiopathic pulmonary fibrosis (Zhang et al., 2011). In the autosomal dominant form of the disease dyskeratosis congenita, for example, a mutation in the gene dyskerin results in reduced amounts of TR in the cell and causes these cells to have shorter telomeres (Wong & Collins, 2003). The significance of the TR was first identified in Tetrahymena where the elongation of telomeres was hindered by the addition of a ribonuclease—an enzyme that degrades RNA. This confirmed the existence of an RNA component of telomerase, and demonstrated that it plays a role in the maintenance of telomeres (Greider & Blackburn, 1987). The first evidence for the existence of a template in the RNA was found in Tetrahymena as well, where single nucleotide mutations made on the template resulted in these nucleotide changes being observed in the telomeres they synthesized (Greider & Blackburn, 1989; Yu et. al, 1990). Other TR template mutations made in yeast revealed a four Senior Thesis to five fold decrease in telomere length in less than 50 generations (Leonardi et al., 2008). In addition to variances in telomere length and repeat sequence, mutations in the Tetrahymena TR template have also demonstrated chromosomal separation abnormalities during cell cycles, resulting in misshapen, abnormal cells (Kirk et al., 1997). These cells that doubled at a significantly slower rate also demonstrated signs of accelerated senescence, as they halted cell division altogether once they reached 18-30 population doublings (Kirk et al., 1997). Early studies done on yeast also confirmed that the telomerase RNA template played a more extensive role than simply serving as the complementary template region for telomere synthesis. TR template mutants had decreased fidelity, as the addition of nucleotides beyond the wild-type template was seen in newly synthesized telomeres (Prescott & Blackburn, 1997). This slippage hinted at the role that TR plays in aligning the template with the telomere end (Prescott & Blackburn, 1997). Investigating telomerase in various model organisms would allow scientists to gain a better understanding of their intricate functions in telomere regulation. The basic biological features and the ease with which the organism can be experimentally manipulated; established filamentous fungi as the ideal organisms for genetic manipulation. However, telomerase and its function in telomere length regulation has not been studied in filamentous fungi (Goldman & Osmani, 2008). Though our lab has been able to demonstrate a decrease in telomere length of TERT mutants in Aspergillus nidulans (Wang, N. et al., 2012), the identification and functional extent of the telomerase RNA in this organism has yet to be established and thus will be the inspiration of my thesis. Aspergillus nidulans as a model organism To facilitate the study of telomeres in eukaryotic cells, our lab utilizes the model organism Aspergillus nidulans. Aspergilli make up group of over 185 filamentous fungi that have a significant impact on modern research (Galagan et al., 2005). The diversity of this family can be seen in the fact that their function can vary anywhere from being a human pathogen to being used in food production and industrial environments (Galagan et al., 2005). Aspergillus nidulans, the most commonly used model organism among them, is a non-pathogenic fungus that undergoes a unique sexual cycle. Coupled with its relatively quick growth and the availability of various wild-type and mutant strains through the Fungal Genetics Stock Center, A. nidulans has been established as a good model for genetic studies (Aspergillus nidulans, December 22, 2009). Healthy colonies of A. nidulans are lush and green when grown in complete media at 37°C. The green color of these colonies comes from the uninucleate asexual spores known as conidia (Figure 2). These spores mature into multinucleate vegetative hyphae that undergo polarized growth (Figure 2). Upon further incubation, these hyphae form conidiophores that yield more conidia, completing their asexual life cycle (Harris et al., 2009) Telomeric research using A. nidulans, began with the identification of its TR repeat sequence 5’-TTAGGG-3’ (Bhattacharyya & Blackburn, 1997). It was also shown that A. nidulans has remarkably short telomeres that average approximately 18 to 19 telomeric repeats per chromosomal end. Recent data from our lab indicates that this short telomere length is also seen in its sexual cells— ascospores (Wang, N. et al., 2012). Attempts to deregulate this short length have been unsuccessful as the length remained stable during assorted growth temperatures, suggesting the organism has a tightly regulated mechanism for the Eukaryon, Vol. 9, March 2013, Lake Forest College maintenance of telomere length (Bhattacharyya & Blackburn, 1997). Mithaq Vahedi ’08 developed a novel approach for effectively measuring telomeres of A. nidulans using an anchored polymerase chain reaction (Wang, N. et al., 2012). This serves as an important tool for the study of short telomeres such as those found in A. nidulans. Using this assay, Vahedi observed that TERT knockout mutations in A. nidulans yield relatively shorter telomeres, indicating the significant role of TERT in telomere regulation in Aspergillus as well (Wang, N. et al., 2012). Similarly, the novel anchored PCR assay may be used to gain a better understanding of the role of the telomerase RNA in A. nidulans that would shed light onto the tight regulatory mechanisms that maintain short telomere lengths in the fungus. Figure 2: Schematic of the asexual life cycle of A. nidulans. Uninucleate asexual spores known as conidia extend a unidirectional germ tube that matures to form multinucleate vegetative hyphae. Hyphae could enter a sexual cycle unique to A. nidulans, or continue in an asexual cycle that forms conidiophores yielding more conidia. Modified from Todd et al., 2007 & Horio, T., 2007. Thesis Aim Our lab has recently identified a putative telomerase RNA candidate in Aspergillus nidulans. This finding has further been supported by Dr. Julian Chen of the University of Arizona (J. Chen, personal communication, May 2011). The goal of my thesis is to mutate this hypothesized telomerase RNA by knocking out the region that serves as a template during telomere synthesis. Based on the results of experiments on other model organisms, I hypothesize that the knockout mutation will cause telomerase to be dysfunctional in the organism. This would confirm whether the hypothesized TR is, in fact, the true TR of A. nidulans. As seen in Tetrahymena and A. nidulans TERT mutants, abnormalities in phenotype are expected due to the deletion of an essential gene that results in dysfunctional telomerase. Using the anchored telomere PCR assay developed in our lab, telomere length studies may be conducted in the TR mutants to gain a better understanding of the tight regulation of short telomeres seen in A. nidulans. Introduction to Thesis Research Gene targeting Gene targeting involves the manipulation of specific regions of the genome, most commonly through a molecular mechanism known as homologous recombination (HR) (Nayak et al., 2006). HR has many functions in cells, and is significant in maintaining the integrity of the genome and avoiding many human cancers (Filippo et al., 2008). This mechanism allows for the pairing of specific regions of two different strands of DNA based on sequence Senior Thesis complementarities. With the aid of enzymes known as recombinases, these regions of DNA can be exchanged (Filippo et al., 2008). Though gene targeting has traditionally required the generation of a DNA construct as a plasmid, novel approaches utilize PCR to construct and amplify enough DNA to be transformed into cells (Goldman & Osmani, 2008). Generation of a DNA Construct Homologous recombination is employed during gene targeting to generate insertions, deletions, or replacements for genetic studies (Nayak et al., 2006). In such gene manipulations, a linear DNA fragment known as a DNA construct is synthesized to replace the wild-type gene with the desired mutation. These DNA constructs are synthesized via PCR using specifically designed primers. Since my thesis attempts to mutate the TR template, I will be deleting the six template nucleotides that are complementary to a telomere repeat and hypothesized to serve as a primer during telomerase-mediated telomere elongation. I designed primers that lacked the template nucleotides to synthesize the DNA construct (Figure 3). These primers, which are intended to induce a deletion mutation, are built complementary to the target DNA and lack hypothesized nucleotides for normal telomerase function. During PCR, the primer anneals to the target DNA, and a polymerase synthesizes a strand complementary to the target DNA. At the end of PCR, linear fragments with the desired mutation— lacking the six template nucleotides — will result. This construct will contain the putative TR template replacement. DNA constructs are usually synthesized in three to four fragments that are then fused together (Figure 4). These fragments are made with complementary flanking regions at their termini that will be significant in allowing them to fuse during the process of “Fusion PCR.” In Figure 4, for example, the 3’ end of Fragment A is complementary to the 5’ end of Fragment B. This allows for Fragments A and B to anneal together at that region, and be amplified as a single fragment by primers that are external to the annealing region. As shown in Figure 4, three such fragments with complementary regions may be fused together in one round of Fusion PCR to result in a DNA construct. The resulting DNA construct is suitable for transformation. It contains the desired mutation and other complementary regions that promote its incorporation into the genome through HR (Figure 4). Studies aimed at maximizing gene-targeting efficiency have indicated that DNA constructs in filamentous fungi need regions of homology at least 500 bp in length for HR to occur (Goldman & Osmani, 2008). Furthermore, knocking out genes that promote DNA damage responses and hinder the possibilities of HR is expected to enhance gene targeting frequency. Figure 3. Design of forward and reverse putative TR template knockout primes. Forward primer (blue) and reverse primer (green) lack a six-nucleotide sequence within the putative TR template of Aspergillus nidulans. The TR template primes the synthesis of the TTAGGG repeat in its telomeres. Ku70 and Ku80 are two such proteins known to play a role in the DNA damage responses initiated via nonhomologous end joining (NHEJ) (Goldman & Osmani, 2008). In Neurospora crassa and other filamentous fungi, there is Eukaryon, Vol. 9, March 2013, Lake Forest College competition between both the NHEJ pathway and HR as both of these systems act upon the transformed molecules, attempting to fix the ends of the introduced DNA (Goldman & Osmani, 2008). Thus, knocking out these Ku proteins results in near exclusive HR that greatly facilitates gene targeting (Goldman & Osmani, 2008). HR of the DNA construct at the desired location occurs at an astonishingly high frequency near 90% in these Ku mutants; however, the 500 bp homologous flanks at both ends of the construct are still required to attain such high levels of integration (Goldman & Osmani, 2008; Nayak et al., 2006). An analysis of the gene targeting efficiency of A. nidulans in Ku mutants confirmed that Ku70 mutants increase gene targeting frequency with little to no effect on growth or sensitivity (Nayak et al., 2006). The TN02A7 strain of A. nidulans used for transformation in my experiments is an auxotrophic Ku70 knockout mutant that was generated by Oakley and is now deposited at the Fungal Genetics Stock Center (Aspergillus nidulans, December 22, 2009). Auxotrophic Mutant Strains and Selectable Markers Successful intake and incorporation of the DNA construct into the genome of the cells being transformed does not always occur. Therefore, it is necessary to select for the cells where transformation has successfully occurred. This is most commonly accomplished by transforming auxotrophic strains. In A. nidulans, for example, auxotrophic strains lack a gene that allows the fungus to synthesize a vital nutrient. These strains do not grow on complete media plates as they usually would unless the lacking nutrient is supplied. Transforming the strains with a DNA construct that contains the desired mutation, as well as the selectable nutritional marker, allows for an efficient way in which transformed cells may be selected. This is because only transformants that have integrated the DNA construct into their genome are able to synthesize the essential nutrients that allow for growth. The TN02A7 strain that will be used in my experiments lacks the nutritional gene pyrG89 (Aspergillus nidulans, December 22, 2009). The lack of this gene in TN02A7 allows for its insertion as a selectable nutritional marker in DNA constructs. My thesis aims to transform a single linear DNA construct that contains the selectable marker for the pyrG89 gene from Aspergillus fumigatus. The pyrG89 gene encodes orotidine 5’- phosphate carboxylase that complements the nutrients uracil and uridine (Osmani et al., 2006). A benefit of using such markers of A. fumigatus is that they have almost identical functions as their complementary genes in A. nidulans. Additionally, the A. fumigatus pyrG89 lacks extensive homology that may prevent their integration into the desired region and cause them to integrate at their complementary auxotrophic marker site instead (Goldman & Osmani, 2008). Protoplast Generation and Transformation Similar to plant cells, fungal cells have a cell wall that serves as a barrier to pathogens and gives these cells their rigid structure. Unlike plant cells, however, these cell walls are largely made of the modified polysaccharide, chitin. During transformation, fungal cell walls need to be degraded to allow for the uptake of the DNA construct through the plasma membrane. Cells that are stripped of their cell walls are known as protoplasts and can be readily identified under phase-contrast microscopy. Typically, spores are inoculated into liquid media and incubated overnight to promote growth of mature germlings and hyphae. These structures are known to convert more easily to protoplasts, unlike ungerminated spores (Osmani et al., 2006). Cell wall degradation is carried out by incubation with enzymes. I used the enzyme Vinoflow® FCE that is Senior Thesis commercially available, as it is used in winemaking and has been shown to have pectinase and beta 1,3-1,6 glucanase activity (Aspergillus nidulans, December 22, 2009). Without cell walls, these multinucleate protoplasts are amazingly resistant to physical shearing and are extremely sensitive to osmotic changes (Osmani et al., 2006; Aspergillus nidulans, December 22, 2009). Polyethylene glycol (PEG) mediated transformation is a commonly used technique that preserves the osmotic balance of protoplasts during transformation and yields great amounts of transformed protoplasts (Kuwano et al., 2008). To induce uptake of the DNA construct, these protoplasts are momentarily incubated in a solution of PEG, other buffers and osmotic stabilizers, and the DNA construct. Though it has been shown that endocytosis is the major mechanism for transformation in mammalian and yeast cells, the process used in filamentous fungi is still unknown (Kuwano et al., 2008). Studies aimed at gaining a better understanding of fungal transformation have concluded that, though PEG facilitates the fusion of protoplasts and increases the frequency with which DNA enters, these two events are independent (Kuwano et al., 2008). I will be using a PEG mediated transformation as it has been proven successful in A. nidulans transformation by several other labs (James, 2011; Osmani et al., 2006). Successful transformants are selected by plating the protoplasts on selective media that lack the nutrient the nutritional marker complements. Since pyrG89 is the selectable marker that allows the synthesis of uracil and uridine, the selective plates used in my thesis experiments will lack these two nutrients thus ensuring the growth of transformants that have integrated the DNA construct containing the pyrG89 gene. Mutating essential genes is lethal, thus successful transformants with the induced mutation are expected to demonstrate an abnormal phenotype that is the result of dysfunctional telomerase. This is especially true for mutations that disrupt telomerase activity, as it is vital for the resetting of telomere length during DNA replication. Phenotypic changes in color, colony size, or a characteristic “spidery” shape are expected due to the loss of telomerase activity caused by a TR template mutation. These phenotypes were observed when A. nidulans TERT mutants were generated by Dr. Peter Mirabito of the University of Kentucky (P. Mirabito, personal communication, 2011,). A fortuitous situation arises because of the ability of A. nidulans to form structures known as heterokaryons that contain two genetically distinct nuclei. Protoplasts are mostly multinucleate, and transformation of the DNA construct into any one of their nuclei could yield transformant growth (Figure 5). This results in heterokaryons that grow in selective media because their untransformed nuclei carry a functional copy of the target gene and other transformed nuclei (depicted in blue) provide the essential selectable marker (Nayak et al., 2006). Thus, these cells don’t display any deficiencies that may arise when an essential gene is mutated because the functional copy of this gene that is present in a second nucleus supplements its need. As asexual conidiophores grow from protoplasts and mature into uninucleate spores, only spores with a transformed nucleus continue to propagate in selective media. To facilitate the propagation and isolation of the desired mutant genotype, scientists use the heterokaryon rescue technique. During the heterokaryon rescue technique, uninucleate conidia from isolated colonies are streaked onto selective and non-selective media plates (Figure 5). If the essential gene is deleted through successful transformation, the colony is expected to demonstrate abnormal growth in selective and non-selective plates. If the essential gene is not deleted and the selectable nutritional marker has not Eukaryon, Vol. 9, March 2013, Lake Forest College Senior Thesis Figure 5: The heterokaryon rescue technique selects for successful mutants. DNA constructs (blue double helices) are transformed into multinucleate protoplasts. Transformed protoplasts may have some nuclei that contain the desired mutation (blue) while other nuclei in the same cell do not (grey). These structures are known as heterokaryons as they consist of two or more genetically distinct nuclei. Asexual conidiophores that mature from these heterokaryon protoplasts produce uninucleate conidia when they sporulate thus spores with the mutation may be isolated. Plating spores on non-selective or selective media can identify mutants with phenotypic deficiencies, as they will grow to the extent possible with a mutated gene (orange check mark). Untransformed spores grow on non-selective media (green check mark) but not on selective media (red “X”) since they lack the DNA construct containing the nutritional marker. been integrated heterologously, no growth will be noticed on the selective plates, and normal growth will be promoted on the non-selective plates. In some cases, the marker integrates heterologously and the desired mutation is not induced. This results in growth on both selective and nonselective plates. The heterokaryon rescue technique will play a vital role in the isolation of mutant transformants and in identifying the phenotype that arises due to the loss of telomerase activity when TR of A. nidulans is knocked out. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.