A telomerase with novel non-canonical roles: TERT controls cellular aggregation and tissue size in Dictyostelium

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect’s rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO’s other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.


Introduction
Each time a chromosome replicates, it loses some DNA from each of its ends. This is not necessarily problematic, because the chromosome is initially capped at each end by a sacrificial strand of non-coding DNA, a telomere [1][2][3]. Further instances of replication, however, can expose the coding DNA, unless the cell can keep repairing the shortened telomeres, by the action of the enzyme complex, telomerase. Accordingly, telomerase, whose main subunits comprise a reverse transcriptase (TERT), and the telomerase RNA component (TERC) [4], has much significance in the biology and pathology of multicellular organisms. As somatic tissues age, for example, telomerase is downregulated, and the resulting telomeric dysfunction can lead to chromosomal instability and various pathologies, including disrupted pregnancies and cancer [5][6][7]. In other cases, the upregulation of telomerase is also associated with, and a biomarker of, some cancers, because it allows the unchecked proliferation of immortalised tumour cells [6,8]. Telomerase also has many non-canonical roles, in which telomere maintenance, or even telomerase activity, is not required [9,10]. For example, telomerase is known to have non-canonical roles in neuronal differentiation [11], RNA silencing [12], enhanced mitochondrial function [13], cell adhesion and migration [14,15] and various cancers [9,16].
Our understanding of telomeres and telomerase began, and has continued to develop, through the study of model organisms such as Drosophila, Zea mays, Tetrahymena, yeast and mice [2,3,[17][18][19][20][21]. One model system in which the possible roles of telomerase have not yet been addressed is Dictyostelium discoideum. This system has proved its usefulness in many contexts, including the study of human diseases [22][23][24][25][26]. One of its advantages is that the processes of cell division (i.e. growth) and development are uncoupled [27], making the organism a highly tractable system for the study, in particular, of differentiation and tissue size regulation [28][29][30][31][32][33][34][35]. In culture, when its bacterial food source is abundant, D. discoideum multiplies as single-celled amoebae. This leads to denser colonies, and exhaustion of the food supply. The rising concentration of a secreted glycoprotein, CMF, triggers the organism to switch to a multicellular mode of development [34,36]. With no resources for further cell proliferation, the amoebae move, in a radial pattern of streams, towards centres of aggregation. Rising levels of secreted proteins, of the counting factor (CF) complex [37,38], trigger a series of changes that lead to breaking up of the streams, which therefore no longer contribute cells to the original aggregate. Each aggregate, which will typically contain 20, 000 to 100,000 cells [39], now rounds up into a mound, which then proceeds through several life-cycle stages, finally forming a spore-dispersing fruiting body about 1-2mm high [34,40]. Mounds can also develop from the breaking-up of a large stream (or aggregate), a process similarly regulated by CF [29,41].
The generic term, 'group', can be used to address the fact that mounds develop from clusters that arise in these slightly different ways, but in this paper we will refer to 'mounds'. Some of the processes and regulators involved in our very abbreviated account of the life-cycle are shown in Fig 1, which focuses on those elements examined in this study.
https://doi.org/10.1371/journal.pgen.1008188.g001 tissue size, in ways analogous to the regulation of tumour size by chalones [42,72]. Third, a putative TERT has been annotated in the D. discoideum genome. It is not known if the RNA component of telomerase (TERC) is present [73] and, in any case, extrachromosomal rDNA elements at the ends of each chromosome in D. discoideum suggest a novel telomere structure [74]. Thus, telomerase in this organism may have a separate mechanism for telomere addition or might have non-canonical roles. As yet, however, there have been no functional studies of TERT reported for D. discoideum.
In this study, we characterize the gene tert in D. discoideum, showing that it has both RT and RNA binding domains. We describe the pattern of tert's expression levels during all stages of development, assay for any canonical telomerase function, and examine the effects of knocking out the gene's function on development. The tert mutant exhibits a wide range of developmental defects, suggesting that wild-type TERT targets multiple elements of the regulatory network depicted in Fig 1. Most interestingly, these defects, and the results of experiments by which we attempt to rescue, or phenocopy, the tert KO phenotype, suggest that this telomerase influences the activity of smlA, and processes downstream of it. Tert thus emerges as one of the upstream genes of the cell-counting pathway, and its overall influence indicates that, despite having no obvious canonical activity, a telomerase can nevertheless play major regulatory roles by virtue of its non-canonical targets.

D. discoideum expresses tert, a gene encoding a protein with telomerase motifs
Extending previous predictions of tert encoding a protein with telomerase motifs [75], our use of the Simple Modular Architecture Research Tool (http://SMART.embl-heidelberg.de) and UniProt (Q54B44) revealed the presence of a highly conserved reverse transcriptase domain and a telomerase RNA binding domain (S1 Fig). These are characteristic of a telomerase reverse transcriptase [76], supporting the idea that the gene we characterized indeed encodes for TERT. The Dictyostelium TERT protein shares 23% and 18.7% identity with human and yeast TERT protein respectively (Pairwise sequence Alignment-Emboss Needle). The protein sequence identities between the TERT of D. discoideum and five other species are tabulated in S1 Table. In the case of the identity with the TERT of humans, the strongest homologies are seen in the reverse transcriptase domain. We did a phylogenetic analysis to examine the relatedness of DdTERT with that of other organisms. For this, TERT amino acid sequences from different organisms were obtained from the NCBI database or Dictybase (http://www. dictybase.org/) or SACGB database (http://sacgb.leibniz-fli.de) and compared with TERT of D. discoideum. Multiple sequence alignment of the TERT amino acid sequences of various organisms including other social amoebae were used to create the phylogenetic tree, employing the MUSCLE alignment feature of MEGAX software [77]. The phylogenetic analysis suggests that D. discoideum TERT falls in a separate clade and is likely to be a distant relative of vertebrate homologs (S2 Fig). The evolutionary history was inferred using the Neighbor-Joining method [78]. The evolutionary distances were computed using the p-distance method and the units shown are the number of amino acid differences per site.
Further, using the fold recognition technique on the I-TASSER server, the structure of D. discoideum TERT was predicted using Tribolium castaneum (telomerase in complex with the highly specific inhibitor BIBR1532; PDB-5cqgA) as a template (S3 Fig). The modeled structure of Dictyostelium TERT also suggests that D. discoideum has a structurally conserved TERT (S3 Fig). Telomerase activity, if any, can be ascertained by performing a Telomeric Repeat Amplification Protocol (TRAP) assay, and activity has been successfully detected in organisms such as humans, C. elegans, yeast, Daphnia, and plants [79][80][81][82][83][84]. However, while human cell lines (HeLa, HEK) did show telomerase activity, we did not detect any telomerase activity in D. discoideum cell extracts (S4 Fig). This concurs with previous findings, namely that the telomeres of D. discoideum have a novel structure [85], and that, in other organisms, TERT has several non-canonical roles [11][12][13].

Constitutive expression of telomerase during growth and development in D. discoideum
In humans, telomerase expression is reported to be low in somatic cells compared to germline and tumour cells [86]. To ascertain if tert expression is differentially regulated during growth and/or development, we performed qRT-PCR using RNA from different developmental stages (0, 4, 8, 10, 12, 16 and 24 h after starvation). Tert expression is higher in development than during growth, (8h and 12 h) (Fig 2), implying that tert plays a prominent role beyond the point at which D. discoideum is responding to starvation. Expression also shows a marked biphasic pattern, with the first peak at 8h (when streams are forming), a big dip during stream breaking (10h) and then rising gradually again to peak at about the time of mound formation (12h).

tert KO leads to delayed development, irregular streaming, and smaller mounds and fruiting bodies
To understand the possible non-canonical roles of tert in development of D. discoideum, tert KO cells generated by homologous recombination were seeded at a density of 5x10 5 cells/cm 2 on non-nutrient buffered agar plates and monitored throughout development. While aggregates appeared by 8 h in the wild-type, and streams began to break at 10 h, in the mutants there was a further 8 h delay before aggregates were seen, and stream breaking began at about 18 h. Because of these delays, 'during aggregation', in this study, refers to 8 h in WT and 16 h in the tert KO, and 'during stream breakup' refers to 10 h in WT and 18 h in the tert KO. Wild-type cells formed long streams of polarized, elongated cells leading to aggregation, but tert KO cells did not form well-defined streams, failing to aggregate even at 5x10 4 cells/cm 2 (wild-type cells aggregated even at a density of 2x10 4 cells/cm 2 ), suggesting an inability to respond to aggregation-triggering conditions (S5 Fig). The mutant's streams were also larger ( Fig 3A). In contrast to streams moving continuously towards the aggregation centre in WT, tert KO streams break while they aggregate (S1 and S2 Videos). They did eventually form aggregates, largely by clumping. During the early stages of aggregate formation, the number of aggregation centres formed by the tert KO was only 10% of that formed by WT (Fig 3B,  p<0.0001). Due to uneven fragmentation, the late aggregates were also of mixed sizes. The tert KO cells did eventually form all of the typical developmental structures, but by the mound stage, continued fragmentation had resulted in the mounds being more numerous, and smaller, on average, than in the WT. This was also the case for fruiting bodies.
Thus, with reference to Fig 1, tert appears to play roles in multiple aspects of Dictyostelium development: the timing of aggregation; streaming; and the regulation of the size of the mound and fruiting body (Table 1A and 1B).

Many processes and regulators are potentially involved in the phenotypic changes of the tert KO
Given the wide-ranging phenotypic defects seen in the tert KO, it seemed likely that tert is one of the key regulators of development in D. discoideum, affecting many of the processes and regulators depicted in Fig 1. We thus monitored the activity or levels of a number of those elements, comparing the wild-type and tert KO (summarised in Table 1A and 1B). As that summary shows, the tert KO showed significant changes from the wild-type in three broad areas: components of the mound-size regulation pathway; cAMP-related processes/regulators; and adhesion-related processes/regulators. As is clear from Fig 1, the factors that influence these features overlap considerably, both in terms of interacting with each other, and in regulating more than one of the various developmental stages disrupted in the tert KO. Telomerase TERT controls tissue size in Dictyostelium Nevertheless, we think it is useful to consider each of them in turn. As we do so below, we describe a series of experiments that largely fall into two broad categories, as shown in summary form in Tables 2 and 3: Those that attempt to rescue the normal phenotype in tert KO cells ( Table 2); and those that attempt to phenocopy, or induce, the tert KO phenotype in wildtype cells (Table 3). First, however, we describe some experiments that support the direct involvement of tert in the effects already noted.

Support for the involvement of tert itself in the tert KO
To support the idea that the changes observed in the tert KO are, in the first instance, due to changes involving tert itself, and not some other factor, we took two approaches: Overexpression of tert, and the use of TERT inhibitors. Most importantly, overexpression of wild-type TERT (act15/gfp::tert) in tert KO cells rescued all three of the phenotypic defects ( Fig 4A, S3 Video; Table 2), suggesting that the tert KO phenotype is not due to any other mutation. Next,  Table 3) and fruiting bodies that were small. The developmental delay, however, was not induced. Since the two inhibitors phenocopied the tert KO to a remarkable degree, it is likely that the inhibitor binding sites of Dictyostelium TERT are conserved. Human TERT [87], which shares a 23% homology with Dictyostelium TERT, failed to rescue the tert KO phenotype (S6 Fig). Surprisingly, the morphologies of TERT-overexpressing lines in the wild-type did not show any significant difference to those of the untreated wild-type ( Fig 4A).
Overall, these results strongly support the idea that the relevant changes in the tert KO involve tert itself. The fact that the TERT inhibitors induced only two of the three tert KO defects is interesting. Given the lack of any apparent interconnection between the pathway that regulates the switch to aggregation, and that regulating mound size, it seems likely that TERT acts on more than one molecular target. It could be that the inhibitors do not perturb that part of TERT that interacts with the target that regulates the switch to development.

Roles of components of the mound size regulation pathway in the tert KO:
smlA, CF, countin and glucose smlA and countin. Compared to the wild-type, in the tert KO cells, smlA and countin expression levels were, respectively, low and high (Fig 5A and 5B; Table 1). Also, Western blots performed with anti-countin antibodies showed higher countin expression in tert KO cells, compared to wild-type ( Fig 5C). When tert was overexpressed in the tert KO background, both countin and smlA expression levels were returned to those of the wild-type (Fig 5A and  5B). This overexpression also rescued all the defects of the tert KO phenotype (Fig 4A;  Table 2). Given the previously proposed regulatory relationship between smlA and countin (Fig 1; [28, 30, 32]), the most parsimonious explanation for the majority of the results reported so far in this study, is that one role of tert in D. discoideum is to promote the expression of smlA, thus indirectly inhibiting countin expression, and thus increasing glucose levels and mound/fruiting body size. This would suggest that tert could be one of the regulators of mound size.
The likelihood of some involvement of CF itself was supported by the effects of antibodies that target its components. When tert KO cells were treated with anti-countin or anti-CF50   antibodies (1:300 dilution), there was a reduction in aggregate fragmentation resulting in larger mounds compared to untreated tert KO controls (Fig 5D; Table 2); the development delay was not rescued. Adding anti-CF45 antibodies did not rescue any of the defects (Fig 5D;  Table 2). Indirect evidence that tert is acting upstream of CF was seen in the lack of effect of adding BIBR 1532 to countin KO cells, which typically exhibit no stream breaking and large mounds [30]. While, as noted above, BIBR 1532 leads to stream breaking and small mounds in wildtype cells, it did not lead to any change in the usual phenotype of countin KO cells (e.g. Fig  6A), which argues against tert acting downstream of countin.
Beyond the observations already noted, a range of other observations support the idea that some of the tert KO's features are due to the increased activity of a secreted mound-size regulating factor, such as countin. Conditioned medium (CM) from tert KO cells induced stream breaking in the wild-type ( Fig 6B; Table 3) and led to reduced mound size. Also, adding tert KO CM to the tert KO itself aggravated the fragmentation phenotype ( Fig 6B; Table 2). Tert KO CM was even capable of inducing stream fragmentation (Fig 6A), and reducing mound size, in countin mutants, suggesting that the CF levels of the tert KO CM were high. In each of these three cases, the tert KO CM not only affected streaming and mound size, but also induced, or aggravated, a development delay (Fig 6A and 6B; Tables 2 and 3). This suggests that the unknown TERT-induced factor that affects the developmental switch is also secreted.
Further, the presence of tert KO cells, even at very low concentrations (10%), was able to partially induce the tert KO phenotype when added to a population of wild-type cells and plated at an overall density of 5x10 5 cells/cm 2 ( Fig 6C; Table 3). The apparent potency of the presumed high CF levels produced by the tert KO cells might partly explain one otherwise unexpected observation: Adding wild-type CM to tert KO cells did not rescue any of their defects ( Fig 6B; Table 2). While the wild-type CM in this case would be expected to act as a diluent of CF (and thus potentially rescue the tert KO), this effect would only be brief. Development occurs over many hours, during which time the tert KO conditions could allow the build-up of CF back to mound-size-limiting levels. Similar reasoning might also explain why CM from countin KO cells (which exhibit undelayed aggregation and normal streaming) did not rescue any of the defects of tert KO cells ( Fig 6A; Table 2).
To determine if TERT plays a similar role in tissue size regulation in other dictyostelids, we checked if tert KO CM also affected the aggregate and mound sizes of other species (D. minutum and D. purpureum, each representing a distinct group in the dictyostelid taxonomy). The CM of tert KO did not affect the aggregate or mound size of the species tested (S7 Fig; Table 3) suggesting that some of the factors regulating mound size may be species specific. The fact that tert KO CM did not show any effect on other dictyostelids suggests that the countin-mediated effect may be species specific.
Glucose rescued streaming and mound size defects, but not the delay. As per the model shown in Fig 1, one of the downstream effects that should be seen if the tert KO has higher levels of CF, is the lowering of glucose levels. Glucose levels during aggregation were measured and in the tert KO were significantly lower (10.7±0.6 mg/ml) compared to wild-type (15.5 ±0.94 mg/ml) (Fig 7A, p = 0.0015). Supplementing 1 mM glucose rescued the aggregate streaming (and mound size), defects of the tert KO (Fig 7B), but not, as expected, the delay ( Table 2).
Antibodies against AprA and CfaD did not rescue the tert KO phenotype. Previous work has shown that deletion of AprA and CfaD genes, involved in a different cell-density sensing pathway to that involving smlA and countin, leads to changes in mound-size [31], but, here, antibodies against AprA and CfaD did not rescue the KO phenotype (S8 Fig), suggesting, again, that impaired mound size determination in the tert KO is largely due to defective CF signal transduction.

Roles of cAMP and cAMP-related processes and factors in the tert KO
Given the perturbations seen in the tert KO, one would predict some abnormalities associated with cAMP dynamics [44-46, [88][89][90]. The role of cAMP in streaming, in particular, has been much studied. Below we examine how various cAMP processes or factors, related to streaming and developmental delay, were affected in the tert KO. Multiple cAMP wave generating centres observed in the tert KO. Starving cells normally aggregate by periodic synthesis and relay of cAMP, resulting in the outward propagation of cAMP waves from the aggregation centres [91]. We visualized cAMP waves by recording the time-lapse of development and then subtracting the image pairs [92]. Coordinated changes in cell shape and movement of cAMP waves can be indirectly visualized by dark field optics because of the differences in the optical density of cells moving/not moving in response to cAMP. Compared to the wild-type, which had a single wave generating centre, the tert KO had multiple wave propagating centres in a single aggregation territory (Fig 8, S9 Fig, S4 and S5 Videos). When the tert KO was rescued by overexpression of wild-type tert, so was the single  wave propagating centre. The optical wave density analysis suggests that cAMP wave propagation is defective in tert KO, also contributing to stream breaking.
cAMP-related gene expression, cAMP levels, chemotaxis and relay were also impaired in the tert KO. Both the switch to aggregation, and normal streaming, require that a great variety of other cAMP-related processes occur properly. We quantified the relative expression of genes involved in cAMP synthesis and signaling in wild-type and tert KO cells by qRT-PCR. With respect to the switch to aggregation, the expression levels of acaA (cAMP synthesis), carA (cAMP receptor), 5'NT (5' nucleotidase), pdsA (cAMP phosphodiesterases), regA and pde4 were low initially but most started to 'recover' closer to the time that the tert KO manages to overcome its developmental delay (Fig 9A-9F). Another, perhaps more meaningful, approach is to compare the levels in the mutant and wild-type when they are at equivalent developmental stages. This was done at two stages (aggregation, stream breaking) for four of the cAMP genes (acaA, carA, pdsA, pde4). During aggregation (i.e. at 8 h in the wild-type; 16 h in the tert KO), acaA and carA expression levels were significantly lower in the mutant, and the other two genes trended lower (Fig 10A). During stream breaking (10 h; 18 h, respectively), only acaA was significantly lower (Fig 10B).
Correspondingly, at 8 h of development, cAMP levels were marginally lower in the tert KO (0.98±0.08 nM in the KO; 1.59±0.15 nM in wild-type; Fig 10C, p = 0.005). By 12 h, however, as the tert KO cells are closer to the time when their streaming will begin (i.e. 16 h) both cAMPrelated gene expression, and cAMP levels increase, implying that the initially down-regulated expression of cAMP signaling might explain the long-delayed switch to aggregation in the tert KO. As to how cAMP-related genes or processes do recover in the absence of TERT, there are no indications in our results, but regulatory networks are well-known to exhibit a degree of robustness [93,94].
As noted, cAMP-related gene expression levels of the tert KO lag behind that of the wildtype, and they increase as the mutant enters a similar developmental phase. When cAMP levels were quantified during aggregation and stream breaking using an ELISA-based competitive immunoassay, the cAMP levels in the wild-type and tert KO were 1.59±0.15 nM and 1.48±0.25 nM, respectively, during aggregation (Fig 10D, p = 0.73); and 1.05±0.11 nM and 0.74±0.70 nM during stream breaking (Fig 10E, p = 0.04). Thus, these lower absolute levels of cAMP in the tert KO may also contribute to abnormal stream breaking, with the amoebae unable to relay signals to their neighbours.
To test whether cAMP-based chemotaxis was normal, we performed an under-agarose chemotaxis assay, towards 10 μM cAMP. The trajectories of cells were tracked and their chemotaxis parameters were quantified. Although the speed of cells towards cAMP was higher in tert KO (16.01±1.39 μm/min) compared to the wild-type (12.74±0.43 μm/min), the directionality was significantly reduced in tert KO cells (0.37±0.03 compared to 0.59±0.04). The chemotactic index of tert KO cells also was lower (0.63±0.05) compared to wild-type cells (0.82±0.06) ( Fig  11A-11C).
The chemotaxis defect of tert KO was not rescued by cAMP pulsing or 8-Br-cAMP. To gain further insights into the streaming defect of the tert KO cells, we examined if cAMP pulsing could rescue the chemotaxis defect [95,96]. cAMP (50nM) pulsing was carried out every 6 minutes for 4 hours and thereafter, the cells were seeded in the starvation buffer at a density of 5x10 5 cells/cm 2 and different developmental stages were monitored (Fig 12A). The streaming defect of tert KO was not rescued by cAMP pulsing, suggesting that other components of cAMP signaling are necessary to rescue the defect. If cAMP receptor activity is compromised, that could also lead to defective signaling and to test this, we used a membrane-permeable cAMP analog 8-Br-cAMP. This has a poor affinity for extracellular cAMP receptors and enters the cells directly [47]. Cells were incubated with 5mM 8-Br-cAMP and after 5 h, the cells were transferred to Bonner's Salt Solution and development was monitored (Fig 12B). If 8-Br-cAMP had rescued the tert KO's defects, this would have suggested an impairment of cAMP receptor function, but this was not observed. Thus, impaired function of the receptor might not be responsible for the tert KO's chemotactic defects. However, it is also possible that the receptor is impaired but retains enough activity to obscure any effects of 8-Br-cAMP.
High adenosine levels in the tert KO induced large aggregation streams. As mentioned previously, adenosine and caffeine are known to alter the cAMP relay [97,98], thereby affecting aggregate size. This occurs in a number of dictyostelids [35]. We observed enhanced expression of 5'NT in the tert KO (Fig 13A, p = 0.0042) suggesting increased adenosine levels (5'NT converts AMP to adenosine). Hence, adenosine levels were quantified and these were significantly higher (235.37±26.44 nM/10 6 cells) in tert KO cells compared to wild-type (35.39 ±12.78 nM/10 6 cells) (Fig 13B, p = 0.0051). The adenosine antagonist, caffeine (1 mM), rescued the streaming defect (Fig 13C), and restored the mound size, suggesting that excess adenosine in the tert KO causes larger streams. It did not, however, rescue the developmental delay. Since glucose also rescues the streaming defect in tert KO cells, adenosine levels were quantified subsequent to treating with 1 mM glucose. Glucose treatment reduced adenosine levels (13.07±7.51 nM/10 6 cells) in tert KO cells to a level that is more comparable to wild-type cells (35.39±12.78 nM/10 6 cells), but as already noted, it did not rescue the developmental delay. Importantly, 5'NT expression and adenosine levels reduced significantly subsequent to stream breaking (S10 Fig). This could perhaps be due to negative feedback on tert itself.
Streaming defects of the tert KO were not due to increased iron levels. Dictyostelium cells, when grown in the presence of 200 μM iron, formed large streams that fragmented into multiple mounds, strikingly resembling the tert KO phenotype [99]. As the phenotypes had similarities, we examined if TERT mediates its effect by altering intracellular iron levels. We quantified iron by ICP-OES and the levels were not significantly different between the wildtype (16.38±1.21 ng/10 7 cells) and tert KO cells (15.25±0.81 ng/10 7 cells) (S11 Fig, p = 0.4573), suggesting that tert KO phenotype is not due to altered iron levels.

The role of adhesion-related factors in the tert KO, as they affect streaming and mound size
Cell-substratum adhesion is also important for migration and proper streaming. By shaking cells at different speeds (0, 25, 50 and 75 rpm), it is possible to vary substratum dependent sheer force. Thus, by counting the fraction of floating cells at different speeds, it is possible to check substratum dependent adhesion. Although both wild-type and tert KO cells exhibited a sheer force-dependent decrease in cell-substratum adhesion, tert KO cells exhibited a significantly weaker cell-substratum adhesion (S12 Fig, p<0.0001), affecting cell motility in a way that might also contribute to stream breaking.
Cell-cell adhesion is also an important determinant of streaming and mound size in Dictyostelium [41]. To examine if adhesion is impaired in the mutant, we checked the expression of two major cell adhesion proteins, cadA, expressed post-starvation (2 h) and csaA expressed during early aggregation (6 h). cadA-mediated cell-cell adhesion is Ca 2+ -dependent and thus EDTA-sensitive, while csaA is Ca 2+ independent and EDTA-resistant [67]. Both csaA and cadA expression were significantly down-regulated (Fig 14A and 14B).
Further, cell adhesion was monitored indirectly by counting the fraction of single cells not joining the aggregate. Aggregation results in the gradual disappearance of single cells and thus it is possible to measure aggregation by determining the ratio of single cells remaining. To examine Ca 2+ -dependent cell-cell adhesion, the assay was performed in the presence of 10 mM EDTA. Both EDTA-sensitive and resistant cell-cell adhesion were significantly defective in tert KO cells (Fig 14C, p = 0.0033 and 14D, p = 0.0015). The levels of csaA and cadA were also lower in the tert KO during aggregation when compared to the WT (Fig 14E, p = 0.0037 and 14F, p = 0.0508). Thus, the delay in tert KO development might be the basis for differences in gene expression.
These results imply that defective cell-substratum and cell-cell adhesion might play roles in the abnormal streaming and mound-size regulation of the tert KO.

The developmental delay of the tert KO was associated with reduced polyphosphate levels
One interesting observation was that the only treatment that fully rescued the tert KO cells was the overexpression of wild-type tert. Also, the only other treatment that rescued the developmental delay itself was mixing wild-type cells with the tert KO cells at a 1:1 ratio (Fig 15;  Table 2). Even though caffeine and glucose rescued streaming and mound size, and apparently this was at least partly mediated via their impact on cAMP-regulated processes, neither of the compounds rescued the delay, even though abnormalities of cAMP-regulated processes are commonly reported causes of delay in other Dictyostelium studies [44-46].
Thus, we examined polyphosphate levels in the tert KO because of their known importance to developmental timing in Dictyostelium [43]. We stained the CM with DAPI for 5 minutes and checked the polyphosphate specific fluorescence using a spectrofluorometer. The CM of tert KO cells has reduced polyphosphate levels (49.55±2.02 μM) compared to wild-type (60.62

Conclusions
Our results reveal that TERT plays an important role in many aspects of Dictyostelium development. The tert KO exhibited a wide range of developmental defects. Despite suitable environmental conditions for multicellular development to begin, the start of the streaming phase is delayed by 8 h. Having once begun, development proceeds and ends abnormally, with large streams, uneven fragmentation, and, eventually, small mounds and fruiting bodies. The wideranging developmental defects are associated with changes to the levels, or expression, of genes and compounds that are known to be highly upstream regulators of the various stages of development, such as streaming and mound/fruiting body formation. Based on the perturbations in the tert KO, and our other experiments, Fig 17 depicts the possible extent of processes, and potential mediating factors, that might depend upon normal tert expression/TERT activity in the wild-type. Note that the arrows that connect tert/TERT to any element in the diagram are not meant to suggest that TERT directly regulates that element, only that TERT is important, perhaps in some indirect way, for the normal levels, or activity, of that element.
One of the most striking findings was that TERT appears to regulate, or is at least necessary for, the normal activity of what was previously known as the most upstream regulator of  mound size, smlA [28,30,32]. Expression levels of smlA were reduced in the tert KO, and we also observed a wide variety of the expected downstream effects of lowered smlA levels. All of these, and a wide variety of treatments that rescued the size-defect of the mutant phenotype, support the idea that the reduction of mound size in the tert KO was indeed mediated via the abnormal functioning of the previously-identified elements of the mound-size regulation pathway.
In addition to the rescue approach, treatments that attempted to phenocopy the tert KO phenotype in the wild-type, also suggest TERT is one of the upstream regulators of mound size. In particular, given that size regulation in D. discoideum depends upon secreted factors of the CF complex, one would have predicted the effects we observed when tert KO CM was added to wild-type cells. Another strong indication that tert acts upstream, at least of CF, was that the inhibition of tert activity in countin mutants failed to phenocopy the tert KO phenotype.
A similarly rich range of results (involving the tert KO phenotype, and its rescue, and phenocopying) support the idea that TERT also plays a high-level role in the regulation of streaming. During the streaming phase, two genes associated with cAMP related-processes in D. discoideum (acaA, carA) were significantly downregulated (compared to the wild-type), and the levels of several other genes trended lower. This was also accompanied by lower cAMP levels. This might explain the defective chemotaxis and cell motility of the tert KO.
Of course, the regulation of streaming is not entirely isolated from that of size. Glucose, one of the central elements of the CF pathway, influences several cAMP-related processes [64]. Thus, it was not surprising that adding 1 mM glucose to the tert KO cells rescued both the size and streaming defects. This study, however, provided a new insight into how the rescue of streaming occurs, because added glucose also reduced adenosine levels. Thus, in the tert KO, the low glucose levels might lead to higher adenosine levels, allowing it to inhibit cAMP related processes (via pathway i, Fig 17). In normal development, given the known sequence of the telomere repeats of D. discoideum (A-G (1)(2)(3)(4)(5)(6)(7)(8) ; [74]), and the fact that telomerase activity would therefore recruit cellular stores of adenosine, it is possible that normal TERT activity keeps adenosine levels low. As yet, however, whether TERT actually acts as a functional telomerase in D. discoideum is not known.
The tert gene we characterized includes the conserved domains and structure of a telomerase reverse transcriptase. Also, supplementing structurally unrelated but specific inhibitors of TERT to wild type cells phenocopies the mutant phenotype. The widely used method to test telomerase activity is the TRAP assay. However, this method failed to detect telomerase activity in D. discoideum and there may be both technical and innate limitations. For example, possible reasons for the lack of any observed activity are that: (i) the presence of rDNA palindrome elements in the chromosomal ends, suggesting a novel telomere structure and the possible role of TERT in maintaining both rDNA and chromosomal termini [74]. This could be an alternate pathway of telomere maintenance in D. discoideum; and (ii) polyasparagine repeats, present in the TERT protein of Dictyostelium, splitting the functional domain into two halves. For telomerase activity, a functional TERT is important in humans [100][101][102]. In yeast as well as humans, truncation of one of the TERT protein domains is known to abolish its function [103,104]. While it is not yet clear whether the apparent absence of canonical TERT function in D. discoideum is due to the absence of normal eukaryote telomeres [105], other studies suggests that TERT is not always associated with telomerase activity. The silkworm genome contains a telomerase gene, but the telomerase itself displays little or no enzymatic activity [106,107]. The telomeres of silkworm consist of the telomeric repeats typical of insects, but also harbor many types of non-LTR retrotransposons [106,108,109]. Also of interest is that species of Calcarea (sponges), Cnidaria (sea anemones and jellyfish) and Placozoa, all have metazoan telomeric sequences, but display little or no telomerase activity [110]. D. discoideum might employ an alternative mode of telomere addition, such as the recombination seen in yeast [111] or the retrotransposition of Drosophila [112,113].
The discussion so far, while it establishes that TERT is needed for several developmental processes to take place, does not help to distinguish whether or not it acts more than once, or if it has more than one target. Could TERT for example act more like the much studied homeodomain proteins, master regulators of animal development, but which only act during very early embryological life [114,115]? Likewise, in D. discoideum, CMF appears to act only once [34]. Two lines of argument suggest that TERT is different.
First, the biphasic nature of tert's expression pattern suggest that it could possibly act during two stages of development. In the wild-type, tert expression builds up to its first peak at 8 h, thus being a potential candidate for enabling streaming to begin, and to proceed correctly, around this time. It then dips markedly to a low point at 10 h, whereby it might help to enable stream break-up by its relative absence. Then, it begins its climb to its second peak at 12 h, when mound size is being finalised. However, it is also possible that the later-occurring defects seen in the tert KO correspond to pleiotropic effects of TERT being absent at a much earlier time-point.
Second, while it is well known that cAMP-related processes play important roles in allowing streaming to begin and to proceed properly, and while we have shown that TERT influences multiple cAMP related processes, the pathway by which TERT influences the initiation of streaming seems distinct from that used for maintaining it. Both glucose and caffeine, for example, rescued the streaming and size defects of the tert KO, but the delay was unaffected. Complementarily, when wild-type cells were mixed at 50% with tert KO cells, they rescued the delay defect only. In fact, the only treatment that fully rescued the tert KO was the overexpression of wild-type tert.
Interestingly, MAP kinase kinase (MEK1) disruption results in a stream-breaking phenotype similar to the tert KO [56], suggesting that MEK1 could be involved in either CF secretion or signal transduction. Also, signals transmitted through p38 mitogen-activated protein kinase (MAPK) regulate hTERT transcription in human sarcoma [116]. We speculate that MEK1 might regulate countin levels through TERT, thus helping to regulate tissue size in D. discoideum.
Also, it is known that MST 312 (a TERT inhibitor) treatment reduces tumour size by 70% in a mouse xenograft model and this inhibition preferentially targets aldehyde dehydrogenasepositive cancer stem cell-like cells in lung cancer [117]. In Dictyostelium, disruption of aldehyde reductase increases group size [118] and, since aldehyde dehydrogenase and aldehyde reductase have opposing activities (oxidation and reduction of aldehydes respectively), they might have opposite functions in group size regulation as well. TERT might possibly be regulating aldehyde reductase activity in determining mound size in D. discoideum.
Other genes are also known to play a significant role in aggregate size determination in Dictyostelium, such as dio3 [119] and pkc [120]. However, it is not known if they interact with TERT in determining mound size.
This study indicates for, the first time, that TERT acts in several non-canonical ways in D. discoideum, influencing when aggregation begins, the processes involved in streaming, and the eventual size of the fruiting body. TERT's influences appear to occur upstream of many other regulators of streaming and fruiting body size. Curiously, as yet we have no evidence that TERT acts as a canonical telomerase, nor is it known whether any other enzyme protects the unusually sequenced telomeres of this species. Given that telomere research is still in progress, we cannot even rule out that TERT's apparently non-canonical roles in D. discoideum development are in fact mediated via some as-yet unidentified action on its unusual telomeres. In the most heavily studied stages of the organism's life-cycle, that is, those that occur in response to starvation, replication has ceased, so further study of this particular point should focus on the amoeboid stage. More generally, this study has revealed a previously unreported non-canonical process influenced by a telomerase, tissue size regulation. This role of TERT, together with its influence on cell motility and adhesion, and the levels of chalone-like secreted factors, bear consideration by those engaged in cancer research.

Dictyostelium culture and development
Wild-type D. discoideum (AX2) cells were grown with Klebsiella aerogenes on SM5 plates, or axenically, in modified maltose-HL5 medium (28.4 g bacteriological peptone, 15 g yeast extract, 18 g maltose monohydrate, 0.641 g Na 2 HPO 4 and 0.49 g KH 2 PO 4 per litre, pH 6.4) containing 100 units penicillin and 100 mg/ml streptomycin-sulphate. Cells were also grown in Petri dishes as monolayers. Other dictyostelid species (D. minutum and D. purpureum) were grown with Klebsiella aerogenes on SM5 plates and cells were harvested when there was visible clearing of bacterial lawns.
To trigger development, cells were washed with KK 2 buffer (2.25 g KH 2 PO 4 and 0.67 g K 2 HPO 4 per liter, pH 6.4) and plated on 1% non-nutrient KK 2 agar plates at a density of 5x10 5 cells/cm 2 in a dark, moist chamber [121]. To study streaming, cells were seeded in submerged condition (KK 2 buffer) at a density of 5x10 5 cells/cm 2 .
BIBR 1532 is a specific non-competitive inhibitor of TERT with IC50 value of 93 nM for human telomerase [122]. To find the optimal dose response of BIBR 1532 in Dictyostelium, starved cells were plated in phosphate buffered agar with different concentrations of BIBR 1532 (10 nM, 25 nM, 50 nM, 100nM and 200 nM) and 100nM was found to be the minimal effective dose in inducing complete stream breaking. MST 312, which is structurally unrelated to BIBR 1532, is a reversible inhibitor of TERT with IC50 value of 0.67 μM for human telomerase [123]. The minimal effective dose in Dictyostelium was found to be 250 nM. Inhibitor treatments were carried out with freshly starved cells resuspended in KK 2 buffer and plated on KK 2 agar plates.

Telomerase activity assay (TRAP)
The TRAP assay takes advantage of the low substrate specificity of telomerase, and involves replacing the telomere sequence with a synthetic template. The telomerase first extends the synthetic substrate primer by adding telomere repeats and these primary products are further amplified by PCR. The primer must have certain modifications, such as an anchor sequence at the 5' end and two mismatches within the telomerase repeats [124,125]. For the TRAP assay in Dictyostelium, we have used different primer sets (S2 Table) according to the basic design principles [124].

Generation of tert knockout (KO) in D. discoideum by homologous recombination
The KO vector for tert disruption was designed following standard cloning procedures. A 5' fragment of 678 bp and a 3' fragment of 322 bp spanning the tert gene (DDB_G0293918) and intergenic regions were PCR amplified and cloned on either side of a bsR cassette in pLPBLP vector (S13 Fig). Restriction endonuclease digestion and DNA sequencing were carried out to confirm the integrity of the KO vector. The tert KO vector was transfected to D. discoideum cells by electroporation. Axenically grown AX2 cells were washed twice with ice-cold electroporation buffer and 1x10 7 cells were resuspended in 100 μl EP ++ buffer containing 10 μg of linearized tert KO vector. The cell suspension mixed with linearized KO vector was transferred to pre-chilled cuvettes (2 mm gap, Bio-Rad) and electroporated (300 V, 2 ms, 5 square wave pulses with 5 s interval) using a BTX ECM830 electroporator (Harvard Apparatus). The cell suspension was then transferred to a Petri dish containing 10 ml of HL5 medium and incubated at 22˚C. After 24 h, the cultures were replaced with fresh HL5 supplemented with 10 μg/ml blasticidin (MP Biomedicals). Blasticidin-resistant clones were screened after three days. Genomic DNA isolated from tert KO clones were subjected to PCR analysis to confirm tert disruption using different primer combinations (S3 Table).

Construction of tert expression vector
Using genomic DNA as template, a 3.8kb tert sequence was PCR amplified using ExTaq polymerase (Takara) and ligated in pDXA-GFP2 vector by exploiting the HindIII and KpnI restriction sites. This vector was electroporated to tert KO and AX2 cells and G418 resistant (10 μg/ ml) clones were selected and overexpression was confirmed by semi-quantitative PCR. Primer sequences used for generating the vectors are mentioned in S4 Table.

Conditioned medium assay
Conditioned medium was prepared as described previously with slight modifications [126]. Briefly, log phase cells of AX2 and tert KO were resuspended at a density of 1x10 7 cells/ml and kept under shaking conditions for 20 h. Cells were pelleted and the supernatant was further clarified by centrifugation. The clarified supernatant (CM) was used immediately. To check the effect of CM on aggregate size, cells were developed in the presence of CM on non-nutrient agar plates and development was monitored. KK 2 buffer was used as control. To deplete extracellular CF with anti-countin antibodies, cells were starved in KK 2 buffer. After 1 h, the cells were developed with anti-countin antisera (1:300 dilution) in KK 2 buffer [65].

Western blot
To examine countin protein expression levels during aggregation, a Western blot was performed with anti-countin antibody. Cells were resuspended in SDS Laemmli buffer, and boiled for 3 min. Subsequently, the samples were run in a 12% SDS-polyacrylamide gel and Western blots were developed using an ECL Western blotting kit (Bio-Rad). Rabbit anti-countin antibodies were used at 1: 3000 dilution.

Cell-cell adhesion assay
Log phase cells were starved at a density of 1x10 7 cells/ml in KK 2 buffer in shaking conditions at 22˚C for 4 h. At the beginning of starvation, 4x10 7 cells were removed and resuspended in 2 ml Sorensen phosphate buffer, vortexed vigorously and 0.4 ml of cell suspension was pipetted immediately in vials containing 0.4 ml ice-cold Sorensen phosphate buffer or 0.4 ml of 20 mM EDTA solution. The cell suspension was then transferred to a shaker and incubated for 30 min and 0.2 ml of 10% glutaraldehyde was added to each sample at the end of incubation and stored for 10 min. Then, 7 ml Sorensen phosphate buffer was added to each vial. Cell adhesion was indirectly measured by counting the number of single cells left behind using a hemocytometer [127].

Cell-substratum adhesion
To measure cell-substratum adhesion, 5x10 5 cells were seeded in 60mm Petri dishes and incubated at 22˚C for 12 h. The Petri dishes with the cell suspension was placed on an orbital shaker at different speeds (0, 25, 50, 75 rpm). After 1 h, adherent and non-adherent cells were harvested, counted using a hemocytometer and the fraction of adherent cells was plotted against the rotation speed [58].

Visualization of cAMP waves
To visualize cAMP wave propagation, 5x10 5 cells/cm 2 were plated on 1% non-nutrient agar plates and developed in dark moist conditions at 22˚C. On a real-time basis, the aggregates were filmed at an interval of 30 s/frame, using a Nikon CCD camera and documented with NIS-Elements D software (Nikon, Japan). For visualizing cAMP optical density waves, image pairs were subtracted [92] using Image J (NIH, Bethesda, MD).

Under agarose cAMP chemotaxis assay
The under agarose cAMP chemotaxis assay was performed as described previously [128]. Briefly, 100 μl of cell suspension starved at a density of 1x10 7 cells/ml in KK 2 buffer was added to outer troughs and 10 μM cAMP was added in the middle trough of a 1% agarose plate. Cells migrating towards cAMP was recorded every 30 s for 15 min with an inverted Nikon Eclipse TE2000 microscope using NIS-Elements D software (Nikon, Japan). For calculating the average velocity, directionality and chemotactic index, each time 36 cells were analyzed. The cells were tracked using ImageJ. Velocity was calculated by dividing the total displacement of cells by time. Directionality was calculated as the ratio of absolute distance traveled to the total path length, where a maximum value of 1 represents a straight path without deviations. Chemotactic index was calculated as the ratio of the average velocity of a cell moving against a cAMP gradient to the average cell speed. It is a global measure of direction of cell motion.

Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from AX2 and tert KO cells at the indicated time points (0-24 h) using TRIzol reagent (Life Technologies, USA) [129]. RNA samples were quantified with a spectrophotometer (Eppendorf) and were also analyzed on 1% TAE agarose gels. cDNA was synthesized from total RNA using cDNA synthesis kit (Verso, Thermo-scientific). 1 μg of total RNA was used as a template to synthesize cDNA using random primers provided by the manufacturer. 1 μl of cDNA was used for qRT-PCR, using SYBR Green Master Mix (Thermo-scientific). qRT-PCR was carried out to analyze the expression levels of tert, acaA, carA, pdsA, regA, pde4, 5'NT, countin and smlA using the QuantStudio Flex 7 (Thermo-Fischer). rnlA was used as mRNA amplification control. All the qRT-PCR data were analyzed as described [130]. The primer sequences are mentioned in S5 Table. cAMP quantification cAMP levels were quantitated using cAMP-XP assay kit as per the manufacturer's protocol (Cell Signalling, USA). AX2 and tert KO cells developed on 1% KK 2 agar, were lysed with 100 μl of 1X lysis buffer and incubated on ice for 10 min. 50 μl of the lysate and 50 μl HRPlinked cAMP solution were added to the assay plates, incubated at room temperature (RT) on a horizontal orbital shaker. The wells were emptied after 3 h, washed thrice with 200 μl of 1X wash buffer. 100 μl of tetramethylbenzidine (TMB) substrate was added and incubated at RT for 10 min. The reaction was terminated by adding 100 μl of stop solution and the absorbance was measured at an optical density of 450 nm. The cAMP standard curve was used to calculate absolute cAMP levels.

Glucose quantification
Glucose levels were quantified as per the manufacturer's protocol (GAHK20; Sigma-Aldrich). Mid-log phase cells were harvested and resuspended at a density of 8x10 6 cells/ml in KK 2 buffer and kept in shaking conditions at 22˚C. Cells were collected again and lysed by freezethaw method. 35 μl of the supernatant was mixed with 200 μl of glucose assay reagent and incubated for 15 min. The absorbance was measured at an optical density of 540 nm. The glucose standard curve was used to calculate absolute glucose levels.

Adenosine quantification
Adenosine quantification was performed as per the manufacturer's protocol (MET5090; Cellbio Labs). Cells grown in HL5 media were washed and seeded at a density of 5x10 5 cells/cm 2 on KK 2 agar plates. The aggregates were harvested using the lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol). 50 μl sample was mixed with control mix (without adenosine deaminase) or reaction mix (with adenosine deaminase) in separate wells and incubated for 15 min. The fluorescence was measured using a spectrofluorometer (Ex-550 nm, Em-595 nm). The adenosine fluorescence in the sample was calculated by subtracting fluorescence of control mixed sample from reaction mixed sample. The adenosine standard curve was used to calculate absolute adenosine levels.

Polyphosphate measurements
The conditioned media was incubated with 25 μg/ml DAPI for 5 min and polyphosphate specific fluorescence was measured using a spectrofluorometer (Ex-415 nm, Em-550 nm) as previously described [131]. Conditioned medium samples were prepared in FM minimal media to reduce the amount of background fluorescence. Polyphosphate concentration, in terms of phosphate monomers were determined using polyphosphate standards.

ICP-OES
ICP-OES was performed as described previously [99]. Cells were developed on KK 2 agar, washed five times in Sorensen phosphate buffer and pelleted. Then, 1 ml of concentrated HNO 3 (70%) was added to each sample, and these were further digested by microwave heating. After digestion, the volume of each sample was brought to 9 ml with ultrapure water, filtered with 0.45 mm filter and analysed by ICP-OES (Perkin Elmer Optima 5300 DV ICP-OES). Sample digestion and metal quantification were carried out at the SAIF facility (Sophisticated Analytical Instrument Facility, IIT Madras).

Microscopy
A Nikon SMZ-1000 stereo zoom microscope with epifluorescence optics, Nikon 80i Eclipse upright microscope or a Nikon Eclipse TE2000 inverted microscope equipped with a digital sight DS-5MC camera (Nikon) were used for microscopy. Images were processed with NIS-Elements D (Nikon) or Image J.

Statistical tools
Microsoft Excel (2016) was used for data analyses. Unpaired Student's t-test and two-way ANOVA (GraphPad Prism, version 6) were used to determine the statistical significance. Development assay at different cell density (2x10 4 cells/cm 2 to 2x10 6 cells/cm 2 ). AX2 cells aggregate even at a cell density below 2x10 4 cells/cm 2 , but tert KO fails to aggregate at such a density. Tert KO phenotype was not rescued even at higher cell density (2x10 6  Cells were plated at a density of 1x10 5 cells/ml, grown overnight, in an orbital shaker. Floating and attached cells were counted and percentage adhesion was plotted versus rotation speed; (n = 3). Both AX2 and tert KO exhibited a sheer force-dependent decrease in substratum adhesion and tert KO exhibited significantly reduced adhesion compared to AX2 cells. (TIF) S13 Fig. Targeted disruption of tert gene (DDB_G0293918) by homologous recombination. A) Physical map of tert gene in the genome. PCR primers are shown at positions where they bind. B) The targeting vector (pLPBLP) with sites of recombination and Blasticidin S resistance gene (Bsr). C) Physical map of the genome after targeted gene disruption. D) PCR amplification of DNA using primers that prime outside the vector (P1 FP) and inside the Bsr cassette (BSR RP); no amplicons were obtained from AX2. E) Amplification of the sequence immediately upstream of the tert gene (P1 FP) and within the tert gene (P2 RP), DNA amplification was observed only in AX2 and not in the tert KO clones. F) PCR of genomic sequences flanking the insertion site. A 3.8 kb fragment from AX2 and 1.5 kb amplicon from the tert KO were observed. G) RT-PCR of tert in the tert KO clone. Ig7 (rnlA) was used as an mRNA amplification control. (TIF) S1  CF45, AprA and CfaD antibodies. Polyphosphate standards were a kind gift from Dr Toshikazu Shiba (RegeneTiss Inc.). hTERT cDNA was a kind gift from Dr Jayakrishnan Nandakumar (University of Michigan). The telomerase activity assay protocol was suggested by Dr Elizabeth Blackburn. We thank Dr Amal Kanti Bera (IIT Madras) for providing HEK and Hela cell lines used in telomerase assay. NN acknowledges Rakesh Mani, Shalini Umachandran, Prajna A Rai and J Meenakshi for discussions. RB wishes to thank Prof. Vidyanand Nanjundiah for his constant support. Geoffrey Hyde acknowledges the advice of Matthew Louttit on figure preparation. This paper is dedicated to the memory of late Prof. John Bonner, Princeton University.