HIF-2α Inhibits Ameloblast Differentiation via Hey2 in Tooth Development.

Enamel is the highly mineralized outer layer of teeth; the cells responsible for enamel formation are ameloblasts. Local hypoxia and hypoxia inducible factor (HIF) in embryonic tissues are important to promote normal organogenesis. However, hypoxic state in tooth germs and the roles of HIF in ameloblast differentiation have not been understood. The aim of this study is to clarify the role of HIF in ameloblast differentiation during tooth germ development. We found that tooth germs were under hypoxia and HIF-1α and HIF-2α were expressed in tooth germs in embryonic mice. Then, we used HIF inhibitors to evaluate the function of HIF during tooth germ development. The HIF-2α inhibitor significantly decreased the size of tooth germs in organ culture, while the HIF-1α inhibitor did not apparently affect the size of tooth germs. The HIF-2α inhibitor enhanced the expression of amelogenin, a marker of ameloblast differentiation, in the tooth germs in organ culture and rat dental epithelial SF2 cells. Moreover, we found that the HIF-2α inhibitor-stimulating amelogenin expression was regulated by hes-related family basic helix-loop-helix transcription factor with YRPW motif 2(Hey2) in SF2 cells. These findings suggest that the HIF-2α-Hey2 axis plays an important role in ameloblast differentiation during tooth germ development.

Talbot interferometry for imaging two-dimensional electron density distribution over discharge plasma with higher sensitivity.

The basic properties of a Talbot interferometer implementing pinhole arrays were experimentally and numerically investigated for the improvement of measurement sensitivity of laser wavefront sensors utilized for electron density imaging over discharge plasmas. A numerical simulation using a plane wave decomposition method indicated that the pinhole arrays with a pitch of 300 µm and a pinhole diameter of 150 µm were most suitable for the measurement of the millimetre-scale discharge plasmas, in consideration of the spatial resolution and measurement accuracy. The plane wave decomposition simulation expected that the measurement sensitivity of the 8th-Talbot-length interferometer could be improved by a factor of 4 compared with the previously developed Shack-Hartmann type laser wavefront sensors, which was experimentally verified by the self-image behavior of the pinhole arrays. The Talbot interferometric system was successfully used for electron density imaging over the vacuum arcs generated between a 3-mm gap. The electron density image observed by the Talbot interferometers was in excellent agreement with that visualized by the previously developed Shack-Hartmann sensors. The practical notification for the pinhole array fabrication was also presented.

A metagenetic approach for revealing community structure of marine planktonic copepods.

Marine planktonic copepods are an ecologically important group with high species richness and abundance. Here, we propose a new metagenetic approach for revealing the community structure of marine planktonic copepods using 454 pyrosequencing of nuclear large subunit ribosomal DNA. We determined an appropriate similarity threshold for clustering pyrosequencing data into molecular operational taxonomic units (MOTUs) using an artificial community containing 33 morphologically identified species. The 99% similarity threshold had high species-level resolution for MOTU clustering but overestimated species richness. The artificial community was appropriately clustered into MOTUs at 97% similarity, with little inflation in MOTU numbers and with relatively high species-level resolution. The number of sequence reads of each MOTU was correlated with dry weight of that taxon, suggesting that sequence reads could be used as a proxy for biomass. Next, we applied the method to field-collected samples, and the results corresponded reasonably well with morphological analysis of these communities. Numbers of MOTUs were well correlated with species richness at 97% similarity, and large numbers of sequence reads were generally observed in MOTUs derived from species with large biomass. Further, MOTUs were successfully classified into taxonomic groups at the family level at 97% similarity; similar patterns of species richness and biomass were revealed within families with metagenetic and morphological analyses. At the 99% similarity threshold, MOTUs with high proportions of sequence reads were identified as biomass-dominant species in each field-collected sample. The metagenetic approach reported here can be an effective tool for rapid and comprehensive assessment of copepod community structure.

Central glial activation mediates cancer-induced pain in a rat facial cancer model.

Peripheral and central glial activation plays an important role in development of pain hypersensitivity induced by inflammation and nerve injury. However, the involvement of glial cells in cancer pain is not well understood. The present study evaluated the peripheral and central glial activation and the effect of an inhibitor of glial activation, propentofylline, on pain-related behaviors in a rat facial cancer model of the growth of Walker 256B cells in the unilateral vibrissal pad until days 3-4 post-inoculation. As compared with sham animals, the facial grooming period was prolonged, the withdrawal latency to radiant heat stimulation was shortened, and the withdrawal threshold by von Frey hair stimulation was decreased at the inoculated region, indicating the development of spontaneous pain, thermal hyperalgesia and mechanical allodynia. In immunostainings for Iba1 and glial fibrillary acidic protein (GFAP), although there were no morphological changes of GFAP-immunopositive satellite glial cells in the trigeminal ganglion, Iba1-immunopositive microglia and GFAP-immunopositive astrocytes in the medullary dorsal horn showed large somata with cell proliferation. After the daily i.p. administration of propentofylline beginning pre-inoculation, the central glial activation was attenuated, the prolonged facial grooming was partially suppressed, and the induced allodynia and hyperalgesia from day 2 were prevented, without a change in tumor size. These results suggest that glial activation in the CNS, but not in the peripheral nervous system, mediates the enhancement of spontaneous pain and the development of allodynia and hyperalgesia at an early stage in the facial cancer model.

Differences between orofacial inflammation and cancer pain.

Rat models of orofacial cancer exhibit both allodynia and hyperalgesia; however, it is unclear whether cancer-induced pain is secondary to cancer-induced inflammation. To address this question, we compared the effects of an anti-inflammatory drug, indomethacin, on pain and neurochemical changes in the medullary dorsal horn in orofacial inflammation and cancer models. Daily peripheral administration of indomethacin largely suppressed mechanical allodynia and thermal hyperalgesia in the inflammation model. The same procedure suppressed allodynia and hyperalgesia in the cancer model, but the suppression was weak when compared with that in the inflammation model. In the medullary dorsal horn, calcitonin gene-related peptide and substance P levels were significantly increased in the inflammation model, but did not change in the cancer model. These results suggest that pain in the orofacial cancer model is not significantly mediated by cancer-induced peripheral inflammation, although it may have some involvement.

Endogenous ghrelin released in response to endothelin stimulates growth hormone secretion in cattle.

The purpose of this study was to evaluate whether circulating ghrelin and growth hormone (GH) concentrations in cattle are regulated by endothelin-1 (ET-1), endothelin-3 (ET-3), and secretin. Six Holstein steers (242+/-1 d old, 280.5+/-4.4 kg body weight [BW]; mean+/-SEM) were allocated randomly in an incomplete Latin square design to receive each of 4 treatment compounds (vehicle, ET-1, ET-3, and secretin) with 1-d intervals between successive treatments. The treatment compounds were injected intravenously via a catheter inserted into the external jugular vein of each steer. Blood was sampled from the indwelling catheter at -30, -15, 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, and 180 min. Plasma ghrelin and GH responses to the treatment compounds were measured by a double-antibody radioimmunoassay system. Data were analyzed by using a MIXED procedure of SAS, version 9.1. Plasma acyl ghrelin, total ghrelin, and GH concentrations were increased by both ET-1 and ET-3 injection (ET-1 injection: 311+/-15 pg/mL vs 245+/-15 pg/mL, 2.4+/-0.2 ng/mL vs 1.61+/-0.05 ng/mL, 4.73+/-0.92 ng/mL vs 1.17+/-0.09 ng/mL for acyl ghrelin, total ghrelin, and GH, respectively; ET-3 injection: 337+/-27 pg/mL vs 245+/-15 pg/mL, 2.6+/-0.1 ng/mL vs 1.61+/-0.05 ng/mL, 5.56+/-0.97 ng/mL vs 1.17+/-0.09 ng/mL for acyl ghrelin, total ghrelin, and GH, respectively; P<0.01). Ghrelin and GH concentrations were not changed by secretin injection throughout the experimental periods. These results indicate that ET-1 and ET-3 stimulate ghrelin and GH secretion in cattle and demonstrate for the first time that endogenous ghrelin released in response to endothelin injection stimulates GH secretion in vivo in cattle.

Serine-arginine-rich nuclear protein Luc7l regulates myogenesis in mice.

Using a gene trap technique, we identified a murine homologue of the yeast LUC7-like gene (Luc7l), which is a serine-arginine-rich protein (SR protein) that localizes in the nucleus through its arginine-serine-rich domain (RS domain) at the C-terminus and shows a speckled distribution pattern. Although its transcripts are widely expressed in embryos and adults, they are rarely detected in adult skeletal muscle, and Luc7l expression was found to be negatively regulated during the course of development of limb skeletal muscle, as well as during in vitro differentiation of the myoblast cell lines Sol8 and C2C12. We also demonstrated that forced expression of Luc7l protein inhibited myogenesis in vitro. Based on our results, Luc7l is thought to play an important role in the regulation of muscle differentiation.

Identification of missense mutation (G365R) of the butyrylcholinesterase (BCHE) gene in a Japanese patient with familial cholinesterasemia.

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the genomic DNA of a 57-year-old Japanese woman who visited our hospital because of pneumonia. The propositus exhibited an unusually low level of BChE activity, whereas her son and daughter had an intermediate level. Immunologically, there was an absence of BChE protein in the propositus's serum. DNA sequence analysis of the propositus demonstrated a point mutation at codon 365 (GGA-CGA), resulting in a Gly-Arg substitution. A family study showed her son and daughter to have the same mutation.

Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5.

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 microM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.

Analysis of amide bond formation with an alpha-hydroxy-beta-amino acid derivative, 3-amino-2-hydroxy-4-phenylbutanoic acid, as an acyl component: byproduction of homobislactone.

In the synthesis of peptidomimetics containing alpha-hydroxy-beta-amino acid, the coupling of this N(beta)-protected beta-amino acid with amine components was generally performed without the protection of its alpha-hydroxyl group. However, the formation of dipeptides in low yield was often observed when sterically hindered amine components were used. Boc-Apns-OH [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid, allophenylnorstatine] (6), which is one of such beta-amino acid derivatives, is intensively employed as a core structure in the development of HIV-1 protease inhibitors. There have been no precise studies, to date, that have examined amide bond formation with alpha-hydroxy-beta-amino acid derivatives as an acyl component. To determine the cause of this low-yield reaction, we studied the amide bond formation focusing on the activation step of N(beta)-protected alpha-hydroxy-beta-amino acid by using a model coupling reaction between 6 and H-Dmt-OR [Dmt: (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid] (7). A significant amount of homobislactone 9 was formed through the activation of the carboxyl group of 6 to the benzotriazole-type active esters such as OBt and OAt. In addition, this homobislactone formation was markedly increased in the presence of a catalytic amount of a base, which exhibited good correlation with the low yield of the amide bond formation, suggesting that homobislactone formation is one major reason for the low yield of the amide bond formation. Moreover, homobislactones were also formed in other derivatives of the N(beta)-protected alpha-hydroxy-beta-amino acid, suggesting a common feature of this type of amino acids. The use of a strong activation method like EDC--HOAt without base addition enhanced amide bond formation, although a small amount of homobislactone may be formed during the coupling reaction.

Chemical studies on antioxidant mechanism of curcumin: analysis of oxidative coupling products from curcumin and linoleate.

As a part of a research project on the antioxidant mechanism of natural phenolics in food components, curcumin, a turmeric antioxidant, was investigated in the presence of ethyl linoleate as one of the polyunsaturated lipids. During the antioxidation process, curcumin reacted with four types of linoleate peroxyl radicals. Six reaction products were observed in the reaction and subsequently isolated. Their structures were determined by physical techniques, revealing that they have novel tricyclic structures, including a peroxyl linkage. On the basis of the formation pathway for their chemical structures, an antioxidant mechanism of curcumin in polyunsaturated lipids was proposed, which consisted of an oxidative coupling reaction at the 3'-position of the curcumin with the lipid and a subsequent intramolecular Diels--Alder reaction.

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line.

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.

Interaction of p130 with, and consequent inhibition of, the catalytic subunit of protein phosphatase 1alpha.

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1alpha (PP1calpha) as a p130-binding protein. The association between p130 and PP1calpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1calpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1calpha also detected the presence of a complex of p130 and PP1calpha. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1calpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1calpha.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

[Necessity of co-operation of the community medical system based on the results from the survey by questionnaires-participation of the home healthcare and/or hospice systems in southern Tama community hospitals].

Home Care Division of Fujisawa Pharmaceutical Co. Ltd. has been providing the local public with the following services: 1) providing aseptic medicines prescribed in the clean room, 2) renting the infusion fluid pumps, and 3) supporting the community cooperation in healthcare services. Last year, we surveyed questionnaires to the public users (patients and caretakers) of these services, in order to understand the actual status of patients after changing from conventional hospitalization to the home infusion therapy (HIT). From the results of our present survey, it was found that the patients and their family members had positively accepted HIT, while 61% of the HIT users exhibited a strong anxiety in their skills and methods of HIT. Moreover, it was also shown that 61% had other means of nursing and treatment in addition to HIT, indicating a great financial burden on the families. Among them, 69% of the HIT users considered that visiting nurses and primary care physicians were the best co-operators, and changed their conventional healthcare system (hospitalization) to HIT. However, the home caretakers showed a high anxiety in their skill in the home healthcare system, specifically HIT, which was generally highly dependent on the medical care, Thus, a good relationship and co-operation with visiting nurses and primary care physicians was one of the major factors for the users to decide to choose HIT instead of their old medical hospitalization. Therefore, in order to make HIT more useful and widely prevail, it is concluded that establishment of the co-operative systems within our local community, where visiting nurses and primary care physicians can easily provide the patients and their family with professional suggestions, advice and actual care whenever the home caretakers need them.

Expression pattern of novel chick T-box gene, Tbx20.

Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, we attempted to isolate the chick H15-related T-box gene and analyze its expression pattern during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression was initially detected in the posterior lateral mesoderm, after which it expanded to the anterior and was intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm.

Primary thyroid lymphoma: diagnosis of immunoglobulin heavy chain gene rearrangement with polymerase chain reaction in ultrasound-guided fine-needle aspiration.

We present a case of primary thyroid lymphoma coexisting with Hashimoto's thyroiditis in a 75-year-old woman in whom B-cell lymphoma was substantiated based on the findings of immunophenotyping and polymerase chain reaction (PCR) gene rearrangement in specimens that had been obtained by ultrasound (US)-guided fine-needle aspiration biopsy (FNAB). The immunophenotyping technique showed A light chain restriction, and PCR-based assays showed a discrete narrow band, which was diagnostic for clonal B-cell proliferation. Analyses of PCR gene rearrangement in US-guided FNAB may be a useful ancillary technique to pathological findings for diagnosis of primary thyroid lymphoma, especially for differentiation between low-grade B-cell lymphomas and Hashimoto's thyroiditis.

The novel G-protein coupled receptor SALPR shares sequence similarity with somatostatin and angiotensin receptors.

A cDNA encoding a novel G-protein coupled receptor (GPCR) was isolated from a human cerebral cortex cDNA library by low stringency hybridization screening. This putative seven-transmembrane domain receptor of 469 amino acids was designated SALPR (Somatostatin- and Angiotensin- Like Peptide Receptor). SALPR shares the highest amount of amino acid similarity with the somatostatin (35% with SSTR5) and angiotensin receptors (31% with AT1). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the SALPR mRNA is predominantly expressed in human brain regions, particularly the substantia nigra and pituitary, although the mRNA can also be detected in the peripheral tissues, albeit at low levels. Chromosomal mapping by radiation hybrid analysis localized the human SALPR gene to the chromosome 5p15.1-5p14. Transient expression of SALPR in COS-1 cells did not produce any binding sites for somatostatin or angiotensin II, indicating the necessity for further study to discover its ligand and physiological significance.

Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity.

The 130-kDa protein (p130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA.

Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.