extfieldsofstudy: Medicine Biology

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A Single Nucleotide Polymorphism Alters the Activity of the Renal Na (cid:1) :Cl Cotransporter and Reveals a Role for Transmembrane Segment 4 in Chloride and Thiazide Affinity*

The thiazide-sensitive Na (cid:1) :Cl (cid:2) cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physi-ological studies on pressure natriuresis and by its in-volvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na (cid:1) :Cl (cid:2) cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 in the oocytes surface colocalizes with the F-404 specific plasma membrane dye and that oocytes injected with EGFP-TSC exhibit significant thiazide-sensitive 22 Na (cid:2) uptake, indicating the EGFP-TSC fluorescence is located in the plasma membrane (24). For densitometry analysis, the plasma membrane fluorescence was quantified by determining the pixel intensity around the entire oocytes circumference using SigmaScan Pro image analysis software. Statistical Analysis— Statistical significance is defined as two-tailed p (cid:6) 0.05, and the results are presented as mean (cid:7) S.E. The significance of the differences between means was tested with the Student’s t test.

The thiazide-sensitive Na ϩ :Cl Ϫ cotransporter (TSC, 1 gene symbol, SLC12A3; locus ID 6559) is the major NaCl transport pathway in the apical membrane of the mammalian distal convoluted tubule and the teleost urinary bladder (1)(2)(3)(4)(5)(6)(7). The fundamental role of the Na ϩ :Cl Ϫ cotransporter encoded by the SLC12A3 gene in preserving the extracellular fluid volume and divalent cation homeostasis has been firmly established by the identification of inactivating mutations of this gene as the cause of Gitelman's disease (8 -10), an inherited disorder featuring arterial hypotension, hypokalemic metabolic alkalosis with hypocalciuria, hypomagnesemia, and renal salt wasting. TSC also serves as the target for the thiazide-type diuretics that are currently recommended as the drug of choice for treatment of hypertension (11). Finally, a defect in TSC regulation by the WNK1 and WNK4 kinases has been implicated in the pathogenesis of a salt-dependent form of human hypertension known as pseudohyopaldosteronism type II (12,13), which features marked sensitivity to hydrochlorothiazide and a clinical picture that is a mirror image of Gitelman's disease (hypertension, hyperkalemia, and metabolic acidosis) (14). Taken together, all these observations suggest that TSC molecular variants, resulting from single nucleotide polymorphisms (SNPs), could contribute to the normal variations in blood pressure in the population at large, to the inherited predisposition toward essential hypertension, and/or to the differential response to diuretic therapy.
Despite the important role of TSC in cardiovascular physiology, pharmacology, and pathophysiology, little is currently known about the structure-function relationships in this cotransporter. By using [ 3 H]metolazone binding to membrane preparations from rat renal cortex, Tran et al. (15) proposed that thiazides and Cl Ϫ share the same binding site. Recent studies in which the functional properties of the cloned cotransporter were determined, however, provided evidence that metolazone competes with both Na ϩ and Cl Ϫ ions (16), suggesting that the thiazide-binding site may be shared by both ions and not only by Cl Ϫ , as suggested by Tran et al. (15). In addition, nothing is known regarding domains or amino acid residues defining the TSC ion transport kinetics or thiazide affinity. So far, within the family of electroneutral cotransporters, some aspects of the structure-function relationships have been investigated only in the two isoforms of the Na ϩ :K ϩ :2Cl Ϫ cotransporter, BSC1/NKCC2 and BSC2/NKCC1. Results between both isoforms, however, have shown important differences suggesting that conclusions reached in one member of the family cannot be extended to the other members. For example, in BSC2/NKCC1, Isenring et al. (17,18) have implicated transmembrane domains 4 and 7 in defining Cl Ϫ transport affinity, whereas recent studies (19 -21) in BSC1/NKCC2 clearly showed that transmembrane domain 2 contains affinity modifier residues for extracellular Cl Ϫ .
In the present study we show that an SNP that changes one amino acid residue in TSC results in a dramatic decrease in TSC function, apparently secondary to a decrease in the intrinsic activity of the cotransporter, and reveals a role of transmembrane segment 4 in TSC affinity for extracellular Cl Ϫ and for thiazide diuretics.

MATERIALS AND METHODS
An extensive search of genome data bases (lpgws.nci.nih.gov/cgi-bin/ GeneViewer.cgi; ncbi.nlm.nih.gov/SNP) was performed to find the SNPs that have been informed within the SLC12A3 gene. The SNPs within the coding regions that were considered as potentially important were incorporated into the rat TSC cDNA by using the QuickChange sitedirected mutagenesis system (Stratagene) following the manufacturer's recommendations. Automatic DNA sequencing was used to corroborate all the mutations. All primers used for mutagenesis were custom-made (Sigma).
Genotyping of the G264A Polymorphism-A restriction fragment length polymorphism method was created for the G264A polymorphism to confirm it and to simplify its detection in 200 normal subjects. Total genomic DNA was extracted from whole blood according to standard procedures. PCR was conducted using 125 ng of genomic DNA using the primer pair sense 5Ј-AGACCGTGCGGGACCTGCTC-3Ј and antisense 5Ј-CCTCCTCCATGGCCTCCTCACCTT-3Ј. PCR was conducted for 34 cycles with denaturation at 96°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, with a final extension step at 72°C for 5 min. The G264A variant was recognized by restriction fragment length polymorphism by using Btg1 (New England Biolabs), and the restriction fragments were separated on 7.5% PAGE, and visualized under ultraviolet light after staining with ethidium bromide. The polymorphism was confirmed by automatic sequencing (AbiPrism®) in all positive cases.
Assessment of the Na ϩ :Cl Ϫ Cotransporter Function-Oocytes were harvested from anesthetized adult female Xenopus laevis frogs, defolliculated, and prepared for microinjection following our standard procedure (16,19). The next day mature oocytes were injected with 50 nl of water or cRNA transcribed in vitro, using the T7 RNA polymerase mMESSAGE kit (Ambion), at a concentration of 0.5 g/l. Oocytes were then incubated for 3 days in ND96 with sodium pyruvate and gentamicin and 1 day in Cl Ϫ -free ND96 (16). The function of the Na ϩ :Cl Ϫ cotransporter was determined by assessing tracer 22 Na ϩ uptake (PerkinElmer Life Sciences) in groups of at least 15 oocytes following our standard protocol (16): 30 min of incubation in a Cl Ϫ -free ND96 medium containing 1 mM ouabain, 0.1 mM amiloride, and 0.1 mM bumetanide, followed by a 60-min uptake period in a K ϩ -free, NaCl medium containing the same drugs plus 2 Ci of 22 Na ϩ per ml. Gluconate was used as a Cl Ϫ substitute and N-methyl-D-glucamine as a Na ϩ substitute. At the end of the uptake period tracer activity was determined for each dissolved oocyte by ␤-scintillation counting.
Western Blotting-Western blot analysis was used to compare WT with mutant protein in cRNA-injected oocytes following our standard protocol (22). In brief, groups of 15 oocytes injected with water or cRNA were homogenized in 2 l/oocyte of homogenization buffer and centrifuged twice at 100 ϫ g for 10 min at 4°C, and the supernatant was collected. Protein extracts from oocytes (four oocytes per lane) were heated in sample buffer containing 6% SDS, 15% glycerol, 0.3% bromphenol blue, 150 mM Tris, pH 7.6, and 2% ␤-mercaptoethanol, resolved by Laemmli SDS-7.5% PAGE, and transferred to a polyvinylidene difluoride membrane. For immunodetection we used a rabbit polyclonal anti-rat TSC antibody (generously provided by Dr. Mark Knepper, National Institutes of Health), diluted 1:1000 (23). The membrane was exposed to anti-TSC antibody diluted in blocking buffer (TTBS, 0.2% Tween 20) overnight at 4°C, subsequently washed in TTBS, and incubated for 60 min at room temperature with alkaline phosphataseconjugated secondary (anti-rabbit) antibody (Bio-Rad) diluted 1:2000 in blocking buffer and washed again. Immunoreactive species were detected using Immun-Star Chemiluminescent Protein Detection Systems (Bio-Rad).
Assessment of the TSC Expression at the Oocytes Plasma Membrane-The surface expression of wild type or mutant TSC (see below) was determined by assessing the fluorescence in the Xenopus oocytes using a TSC fusion construct that we have previously validated (12,24). In this construct, the EGFP was fused to the amino-terminal domain of TSC. Xenopus oocytes were then microinjected with water as control or with EGFP-WT-TSC or EGFP-mutant-TSC cRNA. After 4 days of incubation, oocytes were monitored for EGFP fluorescence in the oocytes surface using a Zeiss laser-scanning confocal microscope (objective lens ϫ10, Nikon). Excitation and emission wavelengths used to visualize EGFP fluorescence were 488 and 515-565 nm, respectively. We have shown previously that EGFP-TSC fluorescence in the oocytes surface colocalizes with the F-404 specific plasma membrane dye and that oocytes injected with EGFP-TSC exhibit significant thiazide-sensitive 22 Na ϩ uptake, indicating the EGFP-TSC fluorescence is located in the plasma membrane (24). For densitometry analysis, the plasma membrane fluorescence was quantified by determining the pixel intensity around the entire oocytes circumference using SigmaScan Pro image analysis software.
Statistical Analysis-Statistical significance is defined as two-tailed p Ͻ 0.05, and the results are presented as mean Ϯ S.E. The significance of the differences between means was tested with the Student's t test.

RESULTS
Single Nucleotide Polymorphisms in the SLC12A3 Gene-Up to 135 SNPs have been informed within the SLC12A3 gene. 127 SNPs are located within intronic sequences and only eight are within exonic sequences. Fig. 1 depicts the proposed TSC topology (25) and the localization of the eight SNPs within the coding sequence. Six SNPs result in no change of the amino acid sequence. These are the SNPs A122A, T465T, S628S, A714A, G875G, and I1017I corresponding to the NCBI SNP cluster IDs rs2304479, rs5801, rs55802, rs5803, rs5804, and rs2289113, respectively. Two SNPs result in a single amino acid change. One is the R863K SNP (cluster ID rs8060046) that was considered as irrelevant because this SNP located within the carboxyl-terminal domain results in a conserved substitution of the positively charged amino acid arginine, which is present in the human cotransporter (8), for the positively charged residue lysine, which is present in the TSC from rabbit, rat, mouse, and fish (2,(25)(26)(27). In contrast, the other SNP that alter the primary sequence of human TSC (ID number rs1529927) predicts a change of the nonpolar amino acid glycine at the position 264 for the residue alanine. The residue glycine is located within the fourth transmembrane domain and is a conserved amino acid residue, not only in the available TSC sequences from rat (25), mouse (27), rabbit (26), human (8), and flounder (2), but also in all members of SLC12 family that include two genes encoding Na ϩ :K ϩ :2Cl Ϫ cotransporters (25,28) and four genes encoding K ϩ :Cl Ϫ cotransporters (29 -31). Thus, the G264A SNP was considered as potentially important and therefore was introduced into TSC by in vitro mutagenesis.
Allele Frequency of the G264A Polymorphism-A restriction fragment length polymorphism strategy was used to assess the presence of G264A SNP by PCR. The PCR product (510 bp) contained a constant Btg1 restriction site, and therefore, when digested, two bands of 461 and 49 bp, respectively, are observed in GG genotype (encoding glycine at position 264 in both alleles). In contrast, in GA genotype heterozygotes (that is one allele encoding glycine and the other encoding alanine at posi- tion 264), a new specific Btg1 restriction site was used, and then four bands of 461, 390, 71, and 49 bp were observed. To test the allele frequency of the G264A polymorphism, 200 Caucasian subjects were genotyped. The sample included 119 males and 81 females with the following characteristics (mean Ϯ S.D.): age 52 Ϯ 16 years, systolic blood pressure 117 Ϯ 11 mmHg, diastolic blood pressure 69 Ϯ 7 mmHg, and body mass index of 24 Ϯ 4 kg/m 2 . None of the subjects had present or past cardiovascular conditions including hypertension, coronary heart disease, stroke, or diabetes. The frequency of the GA genotype was 2% in the sample studied. No subjects with AA phenotype were detected (that is homozygotes encoding alanine at position 264 in both alleles). Shown in Fig. 2A is a representative gel containing the Btg1-digested PCR fragment from a normal subject and one heterozygous for the GA genotype. Fig. 2, B and C, illustrates sequencing of this region in a GG and a GA phenotype (codons GGC and GCC, respectively). Thus, our results suggest that G264A is a true SNP.
Functional Consequences of the TSC G264A Polymorphism-The functional consequences of the G264A SNP were assessed using a heterologous expression system in X. laevis oocytes. This expression system has been shown to be an excellent tool for a robust and reproducible expression of TSC in our hands (2,12,16,24,25,32) and also in other laboratories (13,27,33,34). In contrast, TSC expression in transfected eukaryotic cells has not been successful in many laboratories, including our own. The best expression so far observed in stably transfected eukaryotic cells (MDCK cells) with human TSC cDNA consists of small increase (ϳ25%) over background (35). Thus, X. laevis oocytes were microinjected with cRNA transcribed from wild type TSC (WT) or from TSC harboring the G264A SNP (G264A). As shown in Fig. 3, WT or G264A cRNA injection induced a significant increase in 22 Na ϩ uptake in X. laevis oocytes. However, the increase in G264A-injected oocytes was of a significantly lower magnitude than the increase observed in WT oocytes. Uptake in WT-injected oocytes was 3448 Ϯ 234 pmol oocyte Ϫ1 h Ϫ1 , whereas in G264A-injected oocytes was 1712 Ϯ 366 pmol oocyte Ϫ1 h Ϫ1 (p Ͻ 0.01, n ϭ 20). As shown in Fig. 3, the uptake was due to the TSC activity because a complete inhibition of uptake was observed in the absence of extracellular chloride or in the presence of the thiazide-type diuretic, metolazone. A reduction of a similar magnitude in the G264A activity was consistently observed in every single ex- . The absence of endogenous thiazide-inhibitable 22 Na ϩ uptake in X. laevis oocytes has been shown before (2,16,25). *, p Ͻ 0.01 versus WT cRNA oocytes in control conditions. n ϭ 20 oocytes per bar. B, ethidium bromide-stained agarose gel showing 2 l of 0.5 g/l of WT and G264A cRNA as stated. C, autoradiograms of Western blot analysis of proteins extracted from WT or G264A cRNA-injected oocytes, as stated. The analysis was performed using rabbit polyclonal anti-TSC antibodies. Comparable immunoreactivities are observed in both lanes. periment. In order to ensure that the reduction in 22 Na ϩ uptake was not due to differences in the amount of cRNA injected or in the transcription rate of the protein, cRNA concentration was determined by densitometry of the corresponding bands in the ethidium bromide-stained agarose gel, and the transcribed protein was assessed by Western blot analysis of the injected oocytes. No differences were found between WT and G264A in the amount of cRNA injected (Fig. 3B), as well as in the TSC protein produced by the oocytes (Fig. 3C).
Surface Expression Analysis in EGFP-WT or EGFP-G264A-Because X. laevis oocytes injected with WT or G264A exhibited similar immunoreactive proteins in the Western blot, we reasoned that potential explanations for the reduction in functional expression of the TSC containing the G264A could be a decrease in the amount of the transporter that reaches the plasma membrane, a decrease in the affinity for the cotransported ions, or a decrease in the intrinsic activity of the cotransporter. To study the first possibility, we assessed the surface expression of the WT and G264A proteins by injecting X. laevis oocytes with the cRNA encoding the WT or G264A cotransporters that had been tagged previously with the EGFP. To perform these experiments, we used the EGFP-tagged TSC construct that we have characterized previously (24) in which the EGFP was fused to the amino-terminal domain of TSC. Then the EGFP cDNA was subcloned into the G264A TSC. The cRNA encoding the EGFP-WT or EGFP-G264A was injected into X. laevis oocytes. As shown in Fig. 4A, densitometry of the corresponding cRNA bands in an ethidium bromide-agarose gel ensured that similar amounts of cRNA were injected. Four days after injection, the surface expression was assessed by monitoring the EGFP fluorescence with a confocal microscope. After oocytes were analyzed in the microscope, half of them were used for protein extraction to assess the EGFP-tagged proteins by Western blot, using a rabbit polyclonal antibody against TSC, and the other half was used in a functional expression assay to determine the thiazide-sensitive 22 Na ϩ uptake. Western blot revealed similar immunoreactive bands in proteins extracted from EGFP-WT-or EGFP-G264A-injected oocytes (Fig. 4B), and surface expression analysis revealed similar fluorescence intensity at the surface of oocytes injected with EGFP-WT or EGFP-G264A, respectively (representative images are shown in Fig. 4, C and D). Fig. 4, E and F, illustrates the plasma membrane fluorescence intensity analysis in 30 EGFP-WT-or 30 EGFP-G264A-injected oocytes, and the functional activity was expressed as 22 Na ϩ uptake in the same oocytes, respectively. Although the surface expression of both clones was comparable, the 22 Na ϩ uptake was reduced in G264A-injected oocytes. Thus, the G264A substitution results in a reduction in the cotransporter activity, which does not appear to be due to a decrease in the surface expression rate.
Ion Transport Kinetics-One potential source of reduction in the functional activity of a membrane transporter is a reduction of the affinity for the transported ions or molecules, preventing the cotransporter from reaching maximal transport capacity when incubated in regular uptake solutions. Thus, we assessed the Na ϩ and Cl Ϫ transport kinetics in X. laevis oocytes injected with WT or G264A cRNA. Shown in Fig. 5, A and C, are the Na ϩ transport kinetics analyses in WT and G264A, respectively. The K m values for Na ϩ transport kinetics were 7.6 Ϯ 1.6 and 5.7 Ϯ 1.1 mM in WT and G264A, respectively, with no significant difference between them. Shown in Fig. 5, B and D, are the Cl Ϫ transport kinetic analyses. The apparent K m value for extracellular Cl Ϫ uptake in WT (Fig. 5B) was 6.3 Ϯ 1.1 mM. This value is similar to that reported previously for the wild type TSC (16). In contrast, the apparent K m value in G264A-injected oocytes was 0.89 Ϯ 0.2 mM (p Ͻ 0.001, n ϭ 3). We repeated the same analysis in triplicate using three different batches of oocytes, and the results were similar. Thus, the G264A resulted in a significant increase in Cl Ϫ affinity on the cotransporter. This increase in ion transport affinity, however, does not explain the reduction in the cotransporter activity.
Diuretic Inhibitory Kinetics-Studies in which the kinetic analyses of [ 3 H]metolazone to renal cortex plasma membranes were assessed suggested that chloride and thiazides may com- pete for the same site in the cotransporter (15). By supporting this possibility we have shown that thiazide affinity in TSC is increased when oocytes are incubated in uptake solutions containing low Cl Ϫ concentration (16). Because an increase in Cl Ϫ affinity in the G264A cotransporter was observed, we assessed the dose-response simultaneously on TSC and G264A for the thiazide-type diuretic metolazone in order to determine the thiazide affinity on each cotransporter. As shown in Fig. 6, when uptakes were performed in 96 mM NaCl (closed symbols and solid lines), the IC 50 value for metolazone-induced reduction in 22 Na ϩ uptake was similar between WT and G264A. The IC 50 value in both was ϳ1 ϫ 10 Ϫ6 M, which is similar to the IC 50 value that we have reported previously for rat TSC (16). However, because the K m for extracellular Cl Ϫ in WT TSC is below 10 mM and in G264A below 1 mM, it is possible that competition between chloride and thiazides does not become apparent by using 96 mM of extracellular Cl Ϫ . Thus, we performed the metolazone dose-response inhibitory curve in the same experiment, but using another group of oocytes in which uptake was done using an extracellular Cl Ϫ concentration around the K m values for this ion, that is ϳ6 mM for TSC and 1 mM for G264A (open symbols and dashed lines). As we have shown before (16), the metolazone IC 50 value in the oocytes injected with WT-TSC changed from ϳ1 ϫ 10 Ϫ6 to ϳ3 ϫ 10 Ϫ7 M, suggesting that metolazone binding to the cotransporter is enhanced when extracellular chloride is lower. In oocytes injected with G264A cRNA, the shift to the left was more dramatic than in WT-TSC because metolazone IC 50 changed from ϳ8 ϫ 10 Ϫ7 M in the presence of 96 mM Cl to ϳ1 ϫ 10 Ϫ8 M when extracellular Cl Ϫ concentration was 1 mM. To test the possibility that the G264A SNP not only affects the affinity for thiazides, but also the diuretic inhibitory profile, we also assessed the dose-response to several thiazides. This experiment was performed simultaneously in WT and G264A-injected oocytes. Therefore, each of the thiazide dilutions that were used was the same for both WT and G264A oocytes. As shown in Fig. 7, the thiazide inhibitory profile of polythiazide Ͼ bendroflumethiazide ϭ trichloromethiazide ϭ benzthiazide Ͼ hydrochlorothiazide ϭ chlortalidone was similar between WT and G264A TSC cotransporters, suggesting that the G264A substitution increases the affinity of the cotransporter for thiazides but does not affect the inhibitory profile.
Role of the Conserved Residues in the Transmembrane Segment 4 -As shown in Fig. 8, the alignment analysis of the transmembrane domain 4 in the electroneutral cotransporter family members revealed that Gly-264 is not the only residue that is conserved in all cotransporters. In addition to Gly-264, the residues Asn-258, Arg-261, and Gly-278 are also conserved in all members of the family. Thus we performed similar substitutions of these residues and analyzed the effects upon the functional properties of the cotransporter. As shown in Fig. 9,  D). A and C, Na ϩ dependence of 22 Na ϩ uptake. B and D, Cl Ϫ dependence of 22 Na ϩ uptake. Uptakes were performed with Na ϩ or Cl Ϫ fixed at 96 mM, varying the concentration of the appropriate counterion from 0 to 40 mM, as indicated. Uptakes were also measured in water-injected oocytes (data not shown), and the mean values for the corresponding water groups were subtracted to analyze only the 22 Na ϩ uptake due to each injected cotransporter. Curve fitting was performed using the Michaelis-Menten equation. Data are expressed as uptakes in pmol oocyte Ϫ1 h Ϫ1 , each point represents the mean of at least 15 oocytes. substitution of these residues resulted in different magnitudes of reduction in the TSC activity. The effect was similar between G264A and N258Q (ϳ50% reduction). Further reduction in the activity was observed in the R261L transporter, whereas the G278A substitution resulted in a complete block of the cotransporter activity. To find out if the decrease in activity was associated or not with a decrease in the surface expression of the cotransporters, X. laevis oocytes were injected with similar amounts of cRNA transcribed from the EGFP-tagged wild type TSC or the EGFP-tagged TSC containing the substitutions, and confocal microscopy analysis of the oocytes was performed 4 days later for assessing the fluorescence intensity at the surface. Most interesting, as shown in Fig. 10, there were no significant differences among all groups. Thus, mutations in TSC on any of the conserved residues in the transmembrane segment 4 produce cotransporters in which the intrinsic activ-ity is reduced. Finally, the ion transport kinetics analysis revealed that in TSC harboring the N258Q and R261L substitutions, the K m values for extracellular Na ϩ were 7.3 Ϯ 0.7 and 8.9 Ϯ 2.3 mM, respectively, and for extracellular Cl Ϫ were 3.2 Ϯ 0.7 (p Ͻ 0.05, n ϭ 4) and 3.2 Ϯ 1.6 mM (p ϭ not significant, n ϭ 4), respectively. Thus, no change was observed in the affinity for extracellular Na ϩ , whereas a slight increase was observed in the affinity for Cl Ϫ . DISCUSSION One hundred and thirty five SNPs have been informed along the SLC12A3 gene that encodes the thiazide-sensitive Na ϩ :Cl Ϫ cotransporter of the renal distal convoluted tubule. Only eight of the 135 SNPs are located within exonic sequences, and one of them, the SNP G264A, results in a significant change of a single amino acid residue. We showed that in our population the distribution of this SNP is 98% homozygous for G264G and 2% for heterozygous G264A. This frequency is different from that shown previously by Melander et al. (36) to be ϳ91% for homozygous G264G, ϳ8% for heterozygous G264A, and ϳ1% for homozygous A264A. When expressed in the heterologous system of X. laevis oocytes, G264A exhibited a reduced maximal transport capacity to about 50% that shown in simultaneous experiments with WT-TSC. As shown by Western blot analysis, the lower activity of the TSC harboring the G264A substitution does not appear to be due to reduced translation of the protein because densitometric analysis demonstrated no differences in the amount of TSC protein produced with or without the G264A substitution. The surface image analysis that was assessed by confocal microscopy in X. laevis oocytes injected with EGFP-tagged WT or G264A cRNA revealed that reduced translocation of the cotransporter to the cell surface is not responsible for the lower activity in G264A, because surface expression in the plasma membrane was comparable between EGFP-WT and EGFP-G264A, whereas the 22 Na ϩ uptake experiments performed with the EGFP-WT-or EGFP-G264Ainjected oocytes revealed a significant reduction in TSC activity in the presence of the G264A substitution. Thus, by taking all these data together; we propose that the lower activity in TSC harboring the G264A substitution is due to a reduced ion translocation rate; i.e. due to a decrease in the intrinsic activity of the cotransporter. A similar negative effect on the intrinsic activity or on the receptor signaling capacity has been documented for the SNP I89V and the SNP S268P that occur in the human high affinity choline transporter (37) and in the human -opioid receptor (38), respectively.
The observation in the present study of one SNP in the renal Na ϩ :Cl Ϫ cotransporter that results in reduction of the cotransporter intrinsic activity suggests a number of testable hypotheses. For example, this SNP may be probably less prevalent in hypertensive patients than in normal subjects, or individuals harboring this SNP probably are less sensitive to treatment with thiazide drugs. In addition, it is well known that TSC activity inversely correlates with calcium reabsorption in the distal tubule (39) and that chronic thiazide treatment is associated with increased bone density (40), which is a protective factor against osteoporosis (41,42). Therefore, another hypothesis could be that SNP G264A is less prevalent in patients with osteoporosis than the general population.
One study addressed the genotype frequency distribution on this G264A SNP in 264 normal and 292 hypertensive subjects from Sweden (36), and no difference was observed between normotensive and hypertensive subjects. It is possible, however, that more hypertensive patients will need to be studied in order to reveal the association between hypertension and certain SNPs. For example, Baker et al. (43) have shown that the T594M mutation in the ␤-subunit of the epithelial sodium channel correlates with the development of hypertension only in black patients with low renin hypertension, and Zhu et al. (44) observed that in white patients, but not in black hypertensive subjects, there is a significant association between the intron 2 conversion allele of the aldosterone synthase gene and the development of essential hypertension.
Regardless of the potential role in the disease discussed above, the G264A SNP reveals a role of the transmembrane domain 4 in the affinity for extracellular Cl Ϫ and also for thiazides. We observed that TSC harboring the G264A exhibits an increase in the affinity for extracellular Cl Ϫ , because the apparent K m value for extracellular Cl Ϫ in G264A was almost 10 times lower than in WT. This increase in affinity for Cl Ϫ was specific for this ion because no change was observed in the affinity for extracellular Na ϩ . Glycine is a non-hydrophobic residue at position 264, which is predicted to be located in the TSC putative transmembrane domain 4 and is conserved in all species in which TSC has been sequenced, including human (8), rat (25), mouse (27), rabbit (26), and flounder (2), as well as among all members of the SLC12 family including TSC, two Na ϩ :K ϩ :2Cl Ϫ , and four K ϩ :Cl Ϫ cotransporter isoforms. Previous studies in the basolateral isoform of the Na ϩ :K ϩ :2Cl Ϫ cotransporter (18,45) indicated that transmembrane helix 4 contains affinity-modifying residues for Cl Ϫ translocation. In addition to Gly-264, there are three other residues within the transmembrane domain 4 that are conserved among all members of the SLC12 family (Fig. 8). We observed that substitution of each of these residues resulted in Na ϩ :Cl Ϫ cotransporters with reduced intrinsic activity, and one of them also exhibited increased affinity for extracellular Cl Ϫ . Thus, as has been suggested by Isenring et al. (18) in the Na ϩ :K ϩ :2Cl Ϫ cotransporter BSC2/NKCC1, it is possible that transmembrane segment 4 in TSC also plays a role in defining the affinity for extracellular chloride. The G278A substitution resulted in a non-functional protein, without affecting the surface expression rate, suggesting that this glycine is completely necessary for the cotransporter to reach its functional conformation state.
The structural mechanisms by which glycine substitution in the TSC fourth transmembrane domain produces the observed changes in its functional properties are not clear, but some hypotheses can be proposed. Glycine is an amino acid residue that plays an important structural role, because this residue allows unusual main chain conformations in proteins. This is probably why glycine is one of the amino acid residues that show high proportion of conservation among homologous protein sequences (46). Although not yet studied in cotransporters, it has been shown in several enzymes that some glycine residues can be important to define the protein flexibility because this residue can be part of a hinge (47)(48)(49)(50), and the extent of rigidity or flexibility in a given hinge has been proposed to play a role in the affinity of the protein for its ligand (51). Thus, one possibility is that the presence of glycine at position 264 provides certain flexibility that in TSC might affect both rates of transport and anion binding. Alternatively, because the four residues that we studied, conserved among all members of the SLC12 family, are hydrophilic (two glycines, one asparagine and one arginine), it is possible that these amino acids mainly face the putative translocation pocket in the cotransporter, and the conformational change that results when these residues were substituted could render the putative translocation pocket more accessible to Cl Ϫ ions but with reduced rates of transport. Further studies will be necessary to clarify these issues Previous studies using the binding of tracer [ 3 H]metolazone suggested that Cl Ϫ ions and thiazide diuretics compete for the same site on the cotransporter (15). Supporting this hypothesis, we have shown that in TSC the higher affinity for chloride FIG. 9. Functional expression of WT and TSC harboring the substitutions G264A, N258Q, R261L, or G278A. The 22 Na ϩ uptake was determined in groups of oocytes that were injected with water or with 25 ng of cRNA from the WT or substituted mutants (as stated). Uptake was assessed in control conditions (open bars) or in the presence of 10 Ϫ4 M of the inhibitor metolazone (closed bars). *, p Ͻ 0.01 versus WT cRNA oocytes in control conditions. n ϭ 20 oocytes per bar.
FIG. 10. Surface abundance of EGFP-tagged WT or EGFPtagged TSC harboring the substitutions G264A, N258Q, R261L, or G278A, as stated. Oocytes were injected with corresponding cRNA, and green fluorescence was assessed by confocal microscopy as described under "Materials and Methods." The mean and standard error of fluorescence is shown for each set of eight oocytes per injection in arbitrary units. is accompanied by a higher affinity for thiazide diuretics. On one hand, the affinity for thiazides is shifted to the left when dose-response curves are performed in low extracellular Cl Ϫ concentrations (16,32), and on the other hand, the prevention of glycosylation in rat TSC increases the affinity for both extracellular Cl Ϫ and thiazides (24). In the present study, we observed that G264A substitution produces a dramatic increase in Cl Ϫ affinity, together with an increase in the affinity for the thiazide-type diuretic metolazone. This observation also supports the hypothesis that in TSC, the affinity-modifying residues for Cl Ϫ may also be involved in defining thiazide affinity, increasing the data supporting that anions and diuretics compete for the same site on the cotransporter. Most interesting, while a similar type of competence between Cl Ϫ and loop diuretics was proposed for the Na ϩ :K ϩ :2Cl Ϫ cotransporter based on studies using [ 3 H]bumetanide (52), the functional analysis of chimeras between the shark and human basolateral BSC2/NKCC1 revealed that changes in Cl Ϫ transport kinetics are not accompanied by similar changes in bumetanide affinity (45).
In summary, we report here the functional characterization of a single nucleotide polymorphism in the SLC12A3 gene that encodes the thiazide-sensitive Na ϩ :Cl Ϫ cotransporter. This membrane protein has been implicated in human diseases such as arterial hypertension and osteoporosis, and the pharmacological modulations of its function are currently used for treating or preventing these disorders. The studied SNP is a substitution of a glycine for alanine in the fourth transmembrane domain, which caused a significant reduction in the Na ϩ :Cl Ϫ transport rate, suggesting that people with this SNP could have an allele with reduced function of the cotransporter. In addition to the effect of the SNP upon the TSC activity, the G264A substitution produced an increase in the affinity of the cotransporter for extracellular Cl Ϫ , but not for Na ϩ , that was accompanied by an increase in the affinity for thiazide diuretics. Thus, our study represents the first detailed examination of genetic polymorphism in the SLC12A3 gene and reveals a role of the TSC transmembrane segment 4 in anion and thiazide affinity.


s2fieldsofstudy: Biology Medicine

text:
Relationship between 3p deletions and telomerase activity in non-small-cell lung cancer: prognostic implications

3p deletions and telomerase reactivation are two of the most frequent events described in relation to non-small-cell lung cancer (NSCLC) pathogenesis. Moreover, a number of genes that map on 3p have been proposed as candidates to tumour-suppressor genes of importance in the lung cancer process. In this work, we analysed deletions at different 3p loci in relationship to telomerase activity in 66 NSCLCs obtained from patients who had suffered potentially curative surgery. Also, we evaluated prognostic implications. DNA samples were analysed for 3p deletions using five different polymorphic human dinucleotide repeat DNA markers (D3S1619 at 3p22.2, D3S3623 at 3p22.1, D3S1260 at 3p21.33, D3S3697 at 3p14.3, and D3S3722 at 3p21.2). Telomerase activity was investigated by a TRAP-based method. Possible correlations between the different molecular markers and distributions of disease-free survival were estimated. Our data revealed a significant correlation between telomerase activity and losses of heterozygosity (LOH) on D3S3697 (P=0.040), since all of the tumours showing deletion at this locus were positives for telomerase. Moreover, our results revealed clear associations with poor prognosis of patients, in the case of LOH at D3S1260 and D3S3697 (P=0.005 and 0.005, respectively). According to our data, potential repressors for telomerase may be located in chromosome 3p.

Lung cancer is one of the most lethal types of cancer to acquire, as reflected in a 5-year survival rate of only 14%. The high mortality rate for lung cancer results, at least in part, from the absence of standard clinical procedures for diagnosis of the disease at early and more treatable stages compared to other cancers. Molecular genetic studies have shown that mutations in proto-oncogenes and tumour-suppressor genes (TSGs) are critical in the multi-step development and progression of lung tumours. Inactivation of TSGs is by far the most common mutational event documented during lung tumorigenesis (Wiest et al, 1997). In this context, losses of heterozygosity (LOHs) on the short arm of chromosome 3 have been frequently reported in tumours from different origin, including lung tumours. In fact, allele loss involving chromosome 3p has been described as one of the most frequent and earliest known genetic events in lung cancer pathogenesis and it is thought that this may affect several potential TSG regions (Kok et al, 1997;Wistuba et al, 2000. A number of genes that map on 3p have been proposed as candidates to TSGs of importance in the lung cancer process. One candidate is FHIT at 3p14.2, which undergoes frequent hemizygous and occasional homozygous deletion in lung cancer cells and encodes a dinucleoside hydrolase (Sozzi et al, 1996). Moreover, the reduced expression of the DNA mismatch repair gene hMLH1 (3p21) has been correlated with allelic imbalance on chromosome 3p in non-small-cell lung carcinomas, and an association between hMLH1 reduced expression and nodal metastasis in squamous cell carcinoma of the lung has been observed (Xinarianos et al, 2000). In addition, the 3p21.3 region has also been extensively examined for putative TSGs, particularly at a 600-kb region homozygously deleted in SCLC cell lines (Wei et al, 1996). More recently, a novel gene encoding a 1755-amino-acid polypeptide has been isolated at 3p22-21.3 and aberrant transcription of this gene, designated DLEC1 (deleted in lung and oesophagus cancer 1), may be involved in carcinogenesis of the lung (Daigo et al, 1999).
In the last few years, acquired loss of the entire or parts of the short arm of chromosome 3 has been considered, by a few authors, in relationship to the lack of regulation of telomerase (Ohmura et al, 1995;Horikawa et al, 1998). In renal cell carcinomas, the presence of at least two genes with regulatory function on the expression of telomerase has been suggested (Mehle et al, 1998). Also, deletion analysis of nonrepressed segregant monochromosome 3 hybrids, in human breast cancer cells, indicated two regions on 3p (3p21.3 -p22 and 3p12 -21.1), where telomerase regulator genes may be located (Cuthbert et al, 1999).
Considering that 3p deletions and telomerase reactivation are two of the most frequent alterations described in relation to lung carcinogenesis, and that there is no report examining both alterations in non-small-cell lung cancer (NSCLC), in this work we have analysed LOH at different 3p loci in relationship to telomerase activity in 66 non-small-cell lung carcinomas obtained from patients who had suffered potentially curative surgery. Also, we have evaluated the prognostic implications for both molecular markers.

Patients and tumour samples
In all, 66 freshly resected lung carcinoma samples were obtained from 66 patients who underwent surgery between 1995 and 2000 at the San Carlos Hospital in Madrid. This study was approved by the Ethical Committee from the Hospital, and informed consent from patients was obtained prior to investigation. Of the 66 patients, four were female and 62 were male, with an average age of 63.5379.39 years. The median follow-up period for patients was 24 months (range 3 -61 months). After surgical resection, samples were immediately frozen and were kept at À801C until used. Molecular analyses were performed in samples containing 480% tumour cells. In all cases, nontumour tissues selected from macroscopically normal areas of surgical specimens were used as controls. Tumours were staged pathologically using the tumour node metastasis (TNM) system (Mountain, 1986) and consisted of 26 TNM Stage I tumours, six TNM II tumours, 26 IIIA tumours, seven TNM IIIB tumours, and one TNM Stage IV tumour. Therefore, 58 patients, who had stage I, II or IIIA tumours, were subjected to curative surgery, whereas only a biopsy was taken from patients who suffered from more extensive disease. According to the World Health Organisation criteria, 39 tumours were squamous cell carcinomas (SCC); 26 were adenocarcinomas (AC); and one was large-cell undifferentiated carcinoma (LCUC). The histological classification of tumours was established according to previous criteria (Sobin, 1982). Thus, 17 tumours were well differentiated; 31 moderately; and 18 poorly differentiated.

Analysis of 3p LOHs
Genomic DNA was isolated from tumour and nontumour tissues as described previously (Blin and Stafford, 1976). DNA samples were amplified using polymerase chain reaction (PCR) and analysed for LOH on chromosome 3p using the five different polymorphic human dinucleotide repeat DNA markers (D3S1619 at 3p22.2, D3S3623 at 3p22.1, D3S1260 at 3p21.33, D3S3697 at 3p14.3, and D3S3722 at 3p21.2) previously reported (Cuthbert et al, 1999). Sequences for these markers were obtained from the National Centre for Biotechnology Information (NCBI) molecular databases. The relative positions of DNA microsatellite markers on 3p are indicated in Figure 1. PCR was performed in a Thermocycler (Gene Amp PCR System 2400; Perkin-Elmer, Norwalk, CT, USA) and was carried out in a 20 ml volume containing 100 ng of genomic DNA; 0.5 mM of upstream and downstream primers; 0.2 mM of each deoxynucleoside triphosphate; 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl 2 ; and 0.5 U of Taq Gold DNA polymerase (Ecogen, SRL, Spain). PCR conditions were as follow: initial denaturation at 951C for 10 min; 35 cycles at 951C for 20 s, at 551C for 20 s, and at 721C for 30 s. The final extension was at 721C for 7 min. After amplification, PCR products were resolved on an ABI 377 Sequencer and analysed by Genescan software (Applied Biosystems, USA). For informative cases, allelic loss was scored if one allele was significantly decreased in tumour DNA (450%) compared to the same allele in normal control DNA.

Evaluation of telomerase activity
Telomerase activity in paired normal and tumour tissues was evaluated as previously published (González-Quevedo et al, 2000), using a Telomerase PCR ELISA Kit from Roche (Roche Molecular Biochemicals, Germany). The method is an extension of the original Telomeric Repeat Amplification Protocol (TRAP) (Kim et al, 1994). Briefly, tissue samples were lysed in ice-cold buffer for 30 min. In the first step, a volume of cell extract containing 6 mg of total proteins was incubated with a biotin-labelled synthetic telomerase-specific primer, and under established conditions the telomerase present in cellular extracts adds telomeric repeats (TTAGGG) to the 3 0 end of the primer. Next, in the second step, these elongation products were amplified by PCR using specific primers. An aliquot of the PCR products was denatured, hybridised to a digoxigenin-labelled, telomeric repeat-specific probe, and bound to a streptavidin-coated microtitre plate. The immobilised PCR products were then detected with an antibody against digoxigenin that is conjugated to peroxidase (anti-DIG-POD). Finally, the probe was visualised by virtue of peroxidasemetabolising TMB to form a coloured reaction product, and semiquantified photometrically (450 nm). Considering that the cutoff for TRAP-ELISA negativity corresponds to OD 450 nm o0.2, all samples showing OD 450 nm 40.2 were judged as telomerase activity positive. As positive control, we used an extract of the telomerase embryonic kidney cell line 293, and negative controls were prepared in each case by treating cell extracts with RNase (DNase-free). Moreover, to avoid the effect of Taq polymerase inhibitors present in the tissue extracts, we estimated the activity of telomerase by serial dilutions of each extract as described previously (Kim et al, 1994).

Statistical analysis
Both 3p deletions and telomerase activity were assessed for potential associations with a number of clinicopathologic  3p deletions and telomerase in NSCLC parameters, including patient gender and age, and TNM stage, histology and differentiation grade of the primary tumours. Moreover, possible associations between the telomerase activity of tumours and LOHs at the different markers considered were evaluated. The relationship between categorical variables was assessed using the w 2 test. A P-value o0.05 was judged to be significant. Distributions of disease-free survival (DFS) were estimated with the Kaplan -Meier method, and comparisons were made with log-rank statistics. Results were considered significant for P-values o0.05. For survival analysis, only patients who had undergone potentially curative surgery (patients with TNM stages I -IIIA tumours) were considered. Thus, the number of patients included in the survival study was 58. Analysis was performed using windows SPSS version 11.0 software.

RESULTS
Loss of heterozygosity analyses were performed in 66 cases of NSCLC. In addition, the normal bronchial tissue from each case was tested simultaneously as a control. Five microsatellite markers mapping on chromosome 3p were chosen to evaluate LOH in the DNAs from all of the samples described in Materials and Methods section. According to our data, the heterozygosity rate for DNA markers tested here was an average of 80% or greater.
Allelic losses on 3p were recorded in 66.7% (44 out of 66) of tumours. Results for individual markers revealed that D3S1619 represented the most frequently altered locus since, only considering informative cases, 20 of 61 tumours (32.7%) showed LOH at this chromosome locus. Loss of heterozygosity frequency for the other markers investigated here revealed that 20.7, 20, 24.2, and 24.2% of informative cases showed deletions at D3S3623, D3S1260, D3S3697, and D3S3722, respectively. In relation with clinico-pathological variables, we did not find any significant correlation with the 3p deletions investigated in this work (data not shown).
Telomerase activity was positive in 55 (83.3%) of 66 NSCLC tissue specimens analysed and negative in 11 (16.7%) cases. The relationship between telomerase activity and clinical and pathologic features is summarised in Table 1. The mean ages at surgery, sex, tumour stage, and histology were not different between the two groups. However, telomerase activity was significantly correlated with poor differentiation of cancers (Table 1).
When we studied the possible associations between LOHs on 3p and telomerase reactivation, our data revealed a significant correlation with LOH on D3S3697 (3p14.3) (P ¼ 0.040). In fact, all of the tumours showing LOH at this locus were positives for telomerase activity ( Table 2). As it can be also observed in Table 2, this analysis indicated a borderline association between D3S1619 (3p22.2) or D3S1260 (3p21.33) deletions and positivity for telomerase. Moreover, 100% of samples showing LOH at D3S1260 had been classified in the group of telomerase-positive cases.
Next, we performed a survival analysis for each one of the variables evaluated in this work. Thus, such as it had been previously reported (González-Quevedo et al, 2002), the Kaplan and Meier survival curves for patients with NSCLC demonstrated that patients with telomerase-positive tumours survived for a shorter period than those with telomerase-negative cancers (P ¼ 0.04).
In relation to 3p deletions, survival studies indicated a diminished DFS time in patients affected by tumours showing this molecular abnormality, but differences were not statistically significant (data not shown). Interestingly, when survival impact was evaluated individually for each one of the DNA markers, only considering informative cases, our results revealed clear associations with poor prognosis of patients in the case of LOH at D3S1260 (3p21.33) and D3S3697 (3p14.3) (P ¼ 0.005 and 0.005, respectively) (Figures 2 and 3).

DISCUSSION
Deregulation of telomerase expression has been directly linked to human diseases, including cancer. The expression of telomerase is suppressed in most normal human somatic cells, but is reactivated during tumorigenesis. This reactivation seems to arrest the normal loss of telomeric DNA incurred as somatic cells divide. Since  continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may be an important step in the acquisition of cell immortalisation, which occurs during tumour progression (Counter et al, 1998;Meyerson, 2000). The mechanisms involved in telomerase regulation are still far from being fully established (Cong et al, 2002). Thus, identification of transcriptional repressors and tumour-specific activators of telomerase subunits should have considerable impact on the understanding of cellular senescence and immortalisation. Non-small-cell lung cancer represents one of the most frequent fatal malignancies in the world. Chromosome 3p deletions have been described as one of the most frequent genetic events in lung cancer pathogenesis and it is though that may affect several potential TSG regions (Kok et al, 1997;Wistuba et al, 2000).
During the last few years, several authors have reported potential associations between telomerase reactivation and 3p deletions. Thus, restoration of the cellular senescence program by chromosome 3 has been associated with repression of telomerase function in renal cell carcinoma (Ohmura et al, 1995). Moreover, it has been suggested that a senescence-inducing gene on chromosome 3 controls hEST2/hTERT gene expression either directly or indirectly, and support the notion that hEST2/hTERT is the major determinant of telomerase enzymatic activity in human cells (Horikawa et al, 1998). More recently, a strong repression of telomerase was observed following transfer of human chromosome 3 into human breast cancer cells (Cuthbert et al, 1999). Authors of this work identified two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located.
Considering the importance of telomerase reactivation and 3p deletions in NSCLC pathogenesis and, on the basis of previous reports, we have investigated possible associations between both molecular markers in non-small-cell lung tumours obtained by surgery from patients affected by this pathology. Losses of heterozygosity on 3p have been evaluated analysing microsatellite markers that map on the regions previously reported as important for repressing telomerase (Cuthbert et al, 1999). The validity of the five DNA markers used here for 3p deletion investigation was corroborated. Thus, our data indicated that the heterozygosity rate for these microsatellite markers was an average of 80% or greater in all cases.
Almost 70% of the tested samples showed 3p deletion in at least one of the loci considered. In spite of these results agreeing with the data previously reported by other authors (Mitsudomi et al, 1996), other studies show higher rates of genetic losses in the chromosome 3p region in lung cancer. Thus, Wistuba et al (2000) detected one or more regions of 3p allele loss in nearly all lung cancer cell lines and in 97% of all resected primary lung tumours. According to these authors, 3p deletions progress in frequency and in size with increasing severity of histopathological changes. The differences in 3p LOH incidences could be attributed to the histological characteristics of tumours. Thus, differences in the allelic loss and mutation patterns have been reported previously between squamous and adenocarcinomas, suggesting that more genetic changes accumulate during tumorigenesis in squamous cell carcinomas than in adenocarcinomas (Sato et al, 1994). This fact may be related to smoking damage (Gazdar and Minna, 1997). However, no association between 3p LOH and smoking exposure was detected in our resected lung carcinoma cases.
On the other hand, our results for telomerase indicated that 83.3% of tumours showed positive activity. These incidences are also similar to that obtained in other previous works (Taga et al, 1999). Furthermore, according to our results, this molecular parameter represented a poor prognosis indicator in NSCLC. In fact, all recurrences were detected in the group of patients with positive telomerase tumours.
When the relationship between telomerase reactivation and 3p deletions at the different loci considered in the present study was established, results obtained showed a significant correlation between telomerase activity and LOH at D3S3697 (mapping at 3p14.3). Moreover, borderline associations were found for LOH at D3S1619 or D3S1260 (located at 3p22.2 and 3p21.33, respectively) and telomerase-positive cases. Interestingly, LOH on two of the three DNA markers was present only in telomerase-positive samples (D3S3697 and D3S1260) and, such as it can be observed in the Results section, survival studies demonstrated the importance of LOHs at these levels in poorer patient prognosis.
Therefore, according to our results, the potential repressors for telomerase may be located in chromosome 3p, and to our knowledge this is the first work in which such association is investigated 'in vivo' in NSCLC tumours. Although the nature of 3p genes implicated in negative regulation of telomerase remains unknown, according to our results, possible TSGs may be involved as candidates. In fact, several genes with importance in lung development and bronchial hyperplasia are located near to the chromosome region where D3S3697 maps (3p14.3) (Figure 1). Between these, two suppressor genes have been proposed. Thus,

Disease-free survival
Months after surgery FHIT (at 3p14.2), which undergoes frequent deletion in lung cancer cells and encodes a dinucleoside hydrolase (Sozzi et al, 1996), could be affected by LOH at D3S3697. On the other hand, DUTT1 (3p12 -13), coding for a receptor with a domain structure of the neural-cell adhesion family, could also be considered. This gene is widely expressed and has been implicated in the guidance and migration of axons myoblasts, and leukocytes in vertebrates. Mice experiments demonstrated that deletion of the 3p region where DUTT1 maps was associated with bronchial epithelial abnormalities including hyperplasia (Xian et al, 2001). Moreover, a novel gene has been mapped in 3p22 -21.3 (Figure 1). This gene, called DLEC1 (deleted in lung and oesophagus cancer 1) (Daigo et al, 1999) has been identified as a candidate TSG, and it has been suggested that it may be involved in carcinogenesis of the lung, oesophagus, and kidney. Our results suggest that deletions in D3S1619 or D3S1260 could be related to LOHs affecting this gene.
Finally, in the group of TSGs on 3p that may be considered as candidates to negative regulation of telomerase, RASSF1 would be included (Figure 1). This putative tumour suppressor identified at 3p21.3 has recently been analysed in relation to NSCLC. Silencing of this gene occurs mainly by promoter methylation (Endoh et al, 2003;Kim et al, 2003), but gene deletion has also been described as a potential mechanism that contributes to lung tumorigenesis (Kuroki et al, 2003).
Therefore, results included in this work corroborate 'in vivo' previous data obtained 'in vitro', suggesting the existence on 3p of one or several genes that participate in telomerase-negative regulation. However, according to gene databases, a wide group of genes on 3p could be related to telomerase regulation, and future and exhaustive studies examining deletions affecting different regions of each one of these genes in relation to telomerase activity are necessary in order to elucidate mechanisms involved in telomerase regulation. In fact, identification of telomerase repressors should have considerable impact on the understanding of cellular immortalisation and cellular senescence, as well as on cancer therapeutic based on the use of telomerase inhibitors.


Sgo stabilizes glue and microtubules

![Graphic][1]

Chromosomes float in cells lacking Sgo (right).

Salic/ElsevierSister chromosomes in mitosis must stick together even as robust kinetochore microtubules try to pull them apart, with the resulting tension helping to correct attachment errors. Now, Adrian Salic, Jennifer

During periods of intense neural activity, calcium oscillations are triggered in astrocytes, inducing the cells to release glutamate. Fellin et al. find that this glutamate release stimulates neuronal activity in hippocampal slices. Photolysis of caged Ca 2 ϩ in single astrocytes almost exclusively activates the extrasynaptic NMDA receptors in CA1 pyramidal neurons. Thus, the astrocytes are triggering neural activity by turning on receptors outside of the synaptic junction itself-receptors that are abundant but whose role has been unknown.
Based on electrical activity and fluorescent probes, the team also found that astrocytes induce synchronous responses in multiple neurons. Although they can't yet say whether a single astrocyte is able to activate multiple neurons, they hypothesize that one glutamate release event may be sufficient to reach the dendrites of several neurons.
"Synchrony is central to information processing," Carmignoto says. And because the astrocytes only release glutamate when the surrounding neural activity is high, their activation of several neurons at once may be important in memory formation or long-term potentiation. The coordinated activity also hints that the astrocytes may be involved in the pathophysiology that underlies epilepsy, which is characterized by coordinated waves of hyperactive neurons. Reference: Fellin, T., et al. 2004. Neuron. 43:729-743. A A delayed activation of multiple neurons (right, bottom) is due to astrocyte glutamate release.

Sgo stabilizes glue and microtubules
ister chromosomes in mitosis must stick together even as robust kinetochore microtubules try to pull them apart, with the resulting tension helping to correct attachment errors. Now, Adrian Salic, Jennifer Waters, and Timothy Mitchison (Harvard Medical School, Boston, MA) have found a protein that works on both sides of this glue-and-pull equation.
Using an in vitro assay for proteins that stimulate microtubule formation, the team isolated the human Shugoshin (Sgo) protein. Yeast and fly homologues are essential for mitosis and their loss is known to cause dramatic defects in sister centromere cohesion in meiosis.
In affinity purification experiments, human Sgo brings down tubulin, and the interaction appears to be direct. Sgo localizes to the kinetochore and is degraded by the anaphase-promoting complex before the cell exits mitosis.
Sgo siRNA causes mitotic arrest and failed centromere cohesion; single chromosomes are found scattered over the entire spindle. Any remaining paired kinetochores are closer than normal, indicating a lack of tension, and fluorescently marked kinetochore microtubules turn over much more rapidly. The double function of Sgo as a kinetochore-microtubule stabilizing protein and a cohesion protein is "an interesting twist, but not unbelievable," says Salic. Reference: Salic, A., et al. 2004. Cell. 118:567-578.

Salic/Elsevier
Crouching dragon guards nascent proteins ells contain a wide variety of chaperones to help newly synthesized proteins fold properly and reach their native state. But just how the chaperones operate isn't always clear. Now, Lars Ferbitz, Nenad Ban (Swiss Federal Institute of Technology, Zurich, Switzerland), Elke Deuerling (University of Heidelberg, Germany), and colleagues find that the trigger factor chaperone complexes with the ribosome and provides a protected cradle for newly forming proteins.
Ferbitz et al. resolved a 2.7 Å crystal structure of the E. coli trigger protein and of the NH 2 -terminal domain of the trigger factor bound to the 50S ribosomal subunit of H. marismortui. The tail domain of the trigger factor binds to the ribosome with moderate affinity. The remainder of the protein-the head, back, and arms-loom over the tunnel where polypeptides exit the ribosome, appearing like a dragon shielding its treasure. "We wouldn't have predicted this structure from the linear sequence," says Ferbitz, especially since the protein is only 48 kD in size.
The chaperone creates a hydrophobic space within which nascent proteins can fold, protected from potentially damaging agents, such as proteases. The team hypothesizes that once a domain in the new protein folds, it loses affinity for the hydrophobic surface of the chaperone. The weak interaction between the ribosome and the trigger factor then gives way, releasing the intact domain. Reference: Ferbitz, L., et al. 2004. Nature . doi:10.1038/nature02899. C Trigger factor protects nascent polypeptides as they exit ribosomes.


extfieldsofstudy:
Computer Science
Biology
Medicine

text:
FESD: a Functional Element SNPs Database in human

We have created the Functional Element SNPs Database (FESD) that categorizes functional elements in human genic regions and provides a set of single nucleotide polymorphisms (SNPs) located within each area. In the FESD, the human genic regions were divided into 10 different functional elements, such as promoter regions, CpG islands, 5′-untranslated regions (5′-UTRs), translation start sites, splice sites, coding exons, introns, translation stop sites, polyadenylation signals and 3′-UTRs, and subsequently, all the known SNPs were assigned to each functional element at their respective position. With the FESD web interface, users can select a set of SNPs in the specific functional elements and get their flanking sequences for genotyping experiments, which will help in finding mutations that contribute to the common and polygenic diseases. A web interface for the FESD is freely available at http://combio.kribb.re.kr/ksnp/resd/.

INTRODUCTION
Single nucleotide polymorphisms (SNPs) coupled with highthroughput genotyping technologies have been chosen as the high-density genetic markers for unraveling complex genetic diseases (1). Almost 9 million human SNPs have been deposited into the dbSNP database (2) and many researchers have tried to discover and validate new SNPs. As the number of SNPs deposited in the dbSNP database is increasing exponentially, many researchers who are interested in complex genetic diseases are focusing on candidate gene approach using genebased haplotypes, which are collections of SNPs located throughout the functional regions of candidate genes (3). Since the number of haplotypes within candidate genes is much smaller than the theoretical number of all possible haplotypes, gene-based haplotypes will give us an effective data-reduction mechanism and enable us to uncover the association of haplotypes with many complex diseases.
A set of SNPs required for constructing haplotypes within the candidate genes can be retrieved from public databases, such as dbSNP, UCSC genome browser or Ensembl browser (2,4). However, since these public databases do not provide user-friendly interfaces for manipulating a set of SNPs, biologists who are not familiar with these public databases have had difficulties in searching a set of SNPs and retrieving their flanking sequences required for genotyping experiments.
There has been a similar program called SNPper, a webbased application designed to automate the tasks of retrieving and extracting SNPs from public databases (5). However, SNPper provides the combined information available only in public resources.
In this study, we have created a Functional Element SNPs Database (FESD) categorizing functional elements in human genic regions and providing their flanking sequences required for genotyping experiments. By using the FESD web interface, researchers can select a set of SNPs for gene-based haplotype study.

Obtaining the human genome sequences and gene information
The reference sequences of human genome are obtained from the NCBI database and, subsequently, functional element sequences are extracted from those using sequence boundary information derived from the UCSC database.
There are 16 tracks within Genes and Gene Prediction Tracks in the UCSC database, such as Known Genes, RefSeq Genes, MGC Genes, Ensembl Genes, Genscan Genes and so on. The length and position of each gene track is slightly different. Among the gene tracks, the RefSeq Genes track is produced from the mRNA sequence data that are generated and curated by the NCBI RefSeq project (6). In this study, we have used RefSeq Genes (refGene) track for gene information. Although there are 21 222 refSeq IDs in the refGene track, the positions of 51 refSeq IDs are not determined and 12 refSeq IDs are located at the mitochondrial DNA. Since we are not interested in the mitochondrial DNA, we used only the valid 21 159 refSeq IDs.

Promoter regions
Human promoter sequences are difficult to identify and are poorly annotated in the public databases. We used the Match program, which is a weight matrix-based tool for searching putative transcription factor binding sites in the DNA sequences (7). Match program is closely interconnected and distributed together with the TRANSFAC database, which was developed more than a decade ago to model factor-site interaction (8). The current version of Match program (TRANSFAC Professional 7.4.1) uses 695 matrices and 5401 transcription factors that were collected in the TRANS-FAC database, and therefore, provides the great possibility to search for a great deal of different transcription factor binding sites.
Although the range of transcription factors may be huge, it is reported that most of the human promoters are located within 2 kb upstream of the transcription start site (7). Therefore, the length of input sequences for the Match program is restricted to 2 kb upstream of the transcription start site. To find transcription factor binding sites with the help of Match program, it is very important to choose appropriate options of the cut-off for core and matrix similarity. We used the minimizing the false positive (FP) errors option for cut-offs and chose the vertebrate option for group of matrices.

CpG islands
CpG islands are associated with genes and are typically common near transcription start sites in vertebrates. CpG islands in the UCSC database are predicted by searching the sequence and scoring each dinucleotide. We downloaded the complete tracks and filtered them out only if they were located within the 2000 bp upstream. Only 12 916 refSeq IDs out of 21 159 had CpG islands in their 2000 bp upstream region.  (6). The 5 0 -UTRs are upstream sequences from the CDS start sites, whereas the 3 0 -UTRs are downstream sequences from the CDS stop sites. In this study, coding exons are considered as CDSs within the transcripts, which mean that 5 0 -UTRs are not coding exons. In contrast, introns are nontranscribed sequences from the DNA sequences; therefore, introns can exist between UTRs and coding exons. The start and end sites of translations are the first and the last 3 bp of CDS, respectively.

Splice sites
We considered the first and the last 2 bp in introns as splice sites (9). The positions of introns are obtained from refGene track in the UCSC database. When any SNPs are found within the splice sites, we place them in the database.

Polyadenylation signal sites
There are several software programs that have been developed to detect PASes in human DNA and mRNA sequences. In one of the early studies, Tabaska and Zhang (10) developed a program named Polyadq, which finds PASes using a pair of quadratic discriminant functions. Recently, Legendre and Gautheret (11) developed Erpin program, which uses 2 g position-specific nucleotide acid patterns to analyze 300 bases upstream and downstream region of a candidate PAS.
In this study, we have used the Erpin program to find PASes from 3 0 -UTR region of the gene. The Linux version of Erpin program was downloaded and installed at a local Linux PC. The training set file was also downloaded from the website and the query database that contains only 3 0 -UTR was subtracted from refGene track in the UCSC database. The recommended optimal parameters are as follows: 'erpin polya_signal.epn <input database> 11,7 -umask 11 -umask 11 4 5 6 7 -cut-off 70% 74% -fwd -unifstat'. The firstlevel search is for the hexameric PAS with a cut-off score of 70%, and the second-level search is for the 46 nt region immediately downstream of the PAS with a cut-off score of 74% (11).

Assigning SNPs to each functional element
This analysis used 8 431 426 SNPs from the dbSNP database (build 120). The position of SNPs was obtained from the flat

Flanking sequence search program: SNPflank
The SNPflank database consists of two types of data: complete genome sequence from the RefSeq genome contigs and the positional information of refSNPs from the dbSNP database (12). Both data were completely downloaded from the NCBI ftp site, parsed with Perl scripts, and subsequently imported into a MySQL relational database.

RESULTS AND DISCUSSION
The distribution of SNPs in the genic-intergenic regions Table 1 shows the distribution of SNPs in the genic-intergenic regions of the human genome. The genic region contained 3 446 791 SNPs (40.8%), whereas the intergenic region contained 4 984 635 SNPs (59.2%). There are more SNPs in the intergenic region; however, the density of the intergenic region is slightly lower than the genic region.    Table 2 shows the distribution of SNPs along with the functional element categories. The proportion of SNPs occurring in the promoter, CpG, 5 0 -UTR, start codon, splice site, coding exon, intron, stop codon, PAS and 3 0 -UTR was 3.68, 1.57, 0.37, 0.002, 0.03, 2.26, 90.04, 0.003, 0.002 and 2.03%, respectively. This is because the proportion of SNPs depends on the length of the regions. If we considered the length of each region, the density of SNPs in each functional region would have been 3.269, 2.956, 3.233, 1.119, 1.326, 2.453, 2.847, 1.985, 2.105 and 3.841, respectively. The density of SNPs in the promoter and UTRs was significantly higher than the average density of genome whose density was 2.814. The density of SNPs in the 3 0 -UTR was slightly higher than that in the 5 0 -UTR regions. Among the functional elements, the density of start codon was the lowest at 1.119.
The density of SNPs in the functional elements is depicted in Figure 2. Critical functional elements such as start codon, splice site and stop codon are likely to possess rare SNP density, whereas the density of other functional elements are similar to average density of the genome. Table 3 shows the density of SNPs along with the functional element categories separated by chromosome. According to  Table 3, the density of SNPs in chr7 is significantly higher than that in others and the density of SNPs in chr21 and chr22 is slightly higher than that in others. In contrast, the density of SNPs in chrX and chrY is slightly lower than that in others. These density differences may be attributable to differences in the input SNP/chromosome from the dbSNP database. For example, the NCBI is hosting two versions of chromosome 7.

Searching for genes
The FESD website provides capabilities for searching for genes in several ways: (i) gene name, mRNA accession number and protein accession number; (ii) LocusLink ID and OMIM ID; (iii) chromosome position and band position; (iv) transcription factor name; and (v) disorder or clinical synopsis ( Figure 3A).

Selecting sets of SNPs
Once a gene is searched and submitted to the FESD server, sets of SNPs can be chosen by two different ways ( Figure 3B). First, by using the hyperlinks that present the number of SNPs located within that area, users can get the sets of SNPs ( Figure 3B). Second, users can merge different sets of SNPs by simply clicking checkboxes in the graphic view. When multiple functional elements are selected, only distinct SNPs derived from overlapped elements will be displayed. At the same time, users will also be able to filter the selected sets of SNPs by certain thresholds such as SNP type (bi-allelic or indel), number of hits in genome, average heterozygosity and probability of real SNPs.

Getting flanking sequences of selected SNPs
FESD provide flanking sequences of selected SNPs for genotyping experiments. There are many options for getting flanking sequences such as length range, alternating case and reverse complement change ( Figure 3C). Flanking sequences of multiple SNPs are provided with the FASTA format ( Figure 3D). Details of FESD are given in the online help page.

FUTURE DIRECTIONS
FESD will be updated regularly to reflect the newly discovered SNPs in the dbSNP database and the change of track information in the UCSC database, and also to improve the methods used for categorizing the functional elements. We also plan to give users more options for choosing sets of SNPs and refining them with the help of validation information of SNPs.