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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
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