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Regular Article |
a Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 and hTrp12 contain insertions between previously described exons 9 and 10 that would alter reading frame and produce Trp proteins truncated in the membrane spanning region if expressed. The hTrp3 variant introduces sequence between exons 8 and 9 that would insert 16 amino acids in the C-terminal region of the protein upstream of the calmodulin and inositol 1,4,5-triphosphate receptor interaction domain. hTrp1, hTrp3, and hTrp4 proteins were detected in both pregnant human myometrial and PHM1-41 membranes; a weak band consistent with hTrp6 expression was detected in pregnant human myometrium. These data are consistent with the presence of proteins that could form putative capacitative Ca2+ channels in human myometrium. Control of the activity of these channels may be important for the control of uterine contractile activity.
calcium, parturition, pregnancy, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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q-mediated activation of phospholipase Cß [1, 2]. Activation of phospholipase C increases the hydrolysis of PIP2 to produce diacylglycerol and inositol 1,4,5-trisphosphate (IP3). IP3 binds to a receptor on the endoplasmic reticulum and triggers release of Ca2+ from intracellular stores. Ca2+ entry from the extracellular environment also contributes significantly to the total intracellular free Ca2+ pool and to the refilling of intracellular stores [1, 3, 4]. This refilling is essential for continued contractile activity in response to a stimulus. Mechanisms proposed for regulation of Ca2+ entry include activation of receptor-operated or second-messenger-activated cation channels and activation of voltage-sensitive Ca2+ channels. In addition, GPCR-stimulated IP3 elevation and depletion of Ca2+ intracellular stores as a result of inhibition of smooth endoplasmic reticulum Ca2+-ATPases by agents such as thapsigargin or cyclopiazonic acid trigger Ca2+ entry from the extracellular environment. The terms capacitative and store-operated Ca2+ entry have been used to describe Ca2+ entry into cells that is dependent on the mobilization of intracellular Ca2+ stores [3–5].
The uterus does not exhibit sustained contractile activity in the absence of extracellular Ca2+ [6, 7]. Although L-type Ca2+ channel blockers significantly attenuate contractant-stimulated contractions, there are conflicting data regarding whether oxytocin increases L-type voltage-operated channel activity in myometrial cells [8–10]. In spite of the relative absence of voltage-operated Ca2+ channels in pregnant human myometrial PHM1-41 cells, removal of extracellular Ca2+ significantly decreased the oxytocin-stimulated rise in [Ca2+]i in these cells, and phospholipase C inhibition almost completely attenuated the increase [11]. Moreover, store-operated Ca2+ entry was elicited by thapsigargin in myometrial cells [11]. These data are consistent with a significant contribution from capacitative Ca2+ entry in myometrial cells.
The product of the Drosophila trp gene has been shown to generate an inward current with some properties related to capacitative Ca2+ entry [12]. The mammalian Trp family contains several subfamilies, one of which includes Trp homologs 1–7 [3–5, 13]. Trps1–7 have recognizable transmembrane domains with six transmembrane segments and a putative ion pore region that exhibits considerable homology across proteins. Responses differ somewhat between Trp proteins from different species and between splice variants of a given Trp. In general, overexpressed Trp1 proteins are associated with Ca2+ entry that is nonselective and either stimulated by thapsigargin and GPCRs or constitutively active [14–16]. The human version of mTrp2 is a pseudogene. Trp3, Trp6, and Trp7 are nonselective, are responsive to GPCR activation and, variably, to thapsigargin treatment, and are activated by diacylglycerol [13, 17–22]. Trp3 is also stimulated directly by IP3 receptor [23]. Trp 4 and Trp 5 are ion selective, stimulated by GPCR activation and IP3, and are variably sensitive to thapsigargin [24–27]. Mammalian Trp proteins are thought to form heterotetramers, and it has been postulated that unique channel properties are determined by the molecular nature of these heterotetramers [12, 13].
Before the molecular basis for capacitative Ca2+ entry can be understood in myometrium, the types of Trp proteins expressed must be determined. In this article, we examine capacitative Ca2+ entry in individual PHM1-41 cells and document the expression of several Trp mRNAs and proteins in these cells and in pregnant human myometrium. We also describe some novel alternative splice variants of hTrp1 and hTrp3.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PHM1-41 cell line was derived from late-term pregnant human myometrium [28]. Cells were cultured in 100-mm dishes in Dulbecco minimal essential medium-high glucose (DMEM) with 10% fetal calf serum (FCS), 50 units/ml penicillin, 50 µg/ml streptomycin, 0.1 mg/ml G418 sulfate (Gibco BRL, Gaithersburg, MD), and 2 mM L-glutamine (Sigma, St. Louis, MO) and used at passages 20–25. Myometrial samples from the lower uterine segment at time of elective cesarean section were obtained from women not in labor and were frozen immediately in liquid nitrogen. Patients provided informed consent, as reviewed by the institutional IRB committee.
Measurement of Intracellular Calcium
PHM1-41 cells were plated at 1 x 105 cells/ml on polylysine-coated glass inserts in 35-mm dishes (MatTek, Ashland, MA). Cells were loaded at room temperature for 30–45 min with 5 µM Fura 2-AM (Molecular Probes, Eugene, OR) in fluorescence buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM glucose, pH 7.4). Cells were perfused at 1 ml/min with fluorescence buffer as indicated. Calcium-free buffer included 100 µM EGTA. Changes in Fura 2 fluorescence in individual cells were measured at 340 and 380 nm excitation and 510 nm emission wavelengths in an InCyt2 Im2 imaging system (Intracellular Imaging Inc., Cincinnati, OH). Where indicated, data are expressed as the mean ± SEM for n dishes. In each dish, 
20 cells/dish were examined; the responses were analyzed and averaged.
Messenger RNA Isolation and RT-PCR
The mRNA was isolated from PHM1-41 cells and from uterine myometrium using the FastTrack 2.0 Kit mRNA isolation system (Invitrogen, Carlsbad, CA). First-strand cDNA was prepared from 100 ng mRNA using AMV reverse transcriptase (Promega, Madison, WI) and 100 ng/µl oligo-dT, except for hTrp4, where the specific reverse primer 5'-TCACAATCTTGTGGTCACGTAA-3' was used in the RT reaction (25 µl total volume). The cDNA product was amplified by PCR using the Trp isoform-specific primers listed in Table 1. The general PCR conditions were as follows: 100 pmol/µl forward and reverse primers, 1.5 mM MgCl2, 0.2 mM deoxynucleotide trisphosphate mix (Promega), and 10 U DNA Taq polymerase (Gibco BRL) in a total volume of 50 µl. The cDNA was denatured at 95°C for 1 min, annealed at 55–64°C as indicated in Table 1 for 1.5 min, and extended at 68°C for 1.5 min for 30–35 cycles, followed by an 8-min final extension. The amplified products were separated by electrophoresis on 2% agarose gels, and the DNA bands were visualized by ethidium bromide staining. In control reactions for PCR, template was not added in the reaction mixture. PCR products were cloned into pCR 3.1-Uni mammalian expression vector using the TA cloning kit (Invitrogen). Sequencing was performed by the UT Health Science Center sequencing facility.
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Western Blot Analysis
Human myometrial tissue was homogenized in homogenizing buffer (100 mM Tris-HCl, 1 mM MgCl2, 0.1 mM PMSF, 0.5 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride [AEBSF; Sigma]). The PHM1-41 cells were lysed in homogenizing buffer and sonicated on ice. These mixtures were centrifuged at 3000 x g for 15 min, and the resulting supernatants were centrifuged at 40 000 x g for 1 h. The membrane pellets were suspended in sample buffer (10 mM Tris-HCl, 1 mM EGTA, 250 mM sucrose, 0.1 mM PMSF, 0.5 mM AEBSF, pH 7.3) and stored at -80°C until used. Membrane protein (20 µg for both human tissue and PHM1-41 cells, 5 µg for COS cells, determined by Lowry assay [Bio-Rad, Hercules, CA]) was subjected to sodium dodecylsulfate-polyacylamide electrophoresis in 7.5% gels and transferred to nitrocellulose membrane (Bio-Rad). Immunoblots were probed with affinity-purified IgG antibodies raised against specific Trp peptides (Alomone Labs, Jerusalem, Israel) diluted 1:200 in phosphate-buffered saline with 0.01% Tween-20 and 5% nonfat milk for 2 h at room temperature, washed, and then incubated for 1 h with anti-rabbit IgG antibody (1:3000 dilution) cross-linked with horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ). Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). Where indicated, antibodies (1 µg) were incubated with 1 µg immunizing peptide (Alomone Laboratories) for 1 h at room temperature prior to be used in control immunoblots.
COSM6 cells were grown in DMEM with 8% FCS, 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. For transient transfection, 1 x 106 cells were plated in 10 ml in 60-mm dishes and transfected the following day at 70% confluency with plasmids and TransFast reagent (Promega) according to the manufacturer's recommendations. N-terminal HA-tagged hTrp4 in pcDNA3 (obtained from Dr. J. Putney, NIEHS, NC) or N-terminal X-Press tagged hTrp3, constructed by conventional RT-PCR techniques from PHM1-41 mRNA and inserted into pcDNA6 (Invitrogen) (6 µg) and 15 µl TransFast reagent were used in each dish. Forty-eight hours after transfection, the cells were harvested for membrane preparation and Western blot analysis as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of individual PHM1-41 cells to allow calcium entry before and after stimuli that provoke capacitative calcium entry was determined. In cells perfused with fluorescence buffer containing 100 µM EGTA, basal [Ca2+]i was 86 ± 5 nM (n = 15). As shown in representative tracings in Figure 1, A–C, addition of 1 mM Ca2+ did not change basal [Ca2+]i. After reperfusion in fluorescence buffer containing 100 µM EGTA, exposure of the same cells to 100 nM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, resulted in a prolonged rise in [Ca2+]i that gradually returned to baseline. Following addition of 1 mM extracellular Ca2+, there was a marked increase in [Ca2+]i. This response to a second addition of extracellular Ca2+ did not occur in cells that were not treated with thapsigargin (data not shown). Essentially 100% of the 114 cells examined in this way demonstrated this elevation of [Ca2+]i in response to intracellular store depletion.
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Capacitative Ca2+ entry can also be stimulated by G-protein-coupled receptor activation. Representative responses of PHM1-41 cells to 100 nM oxytocin are shown in Figure 1, D–F. When perfused in fluorescence buffer containing 100 µM EGTA, 87% ± 3% (130/149) of the cells (in a total of seven dishes) examined responded to oxytocin with a significant increase in [Ca2+]i. After [Ca2+]i returned to basal levels, 1 mM Ca2+ was re-added to the medium and 62% ± 6 % (80/130) of the cells responded with a second increase in [Ca2+]i. The magnitude of the second increase in [Ca2+]i was variable: 73% ± 8% (59/80) with a small but detectable increase in [Ca2+]i (Fig. 1D) and 26% ± 8% (21/80) of the cells responded with a large increase in [Ca2+]i, as illustrated in Figure 1E. We have never observed a second increase in [Ca2+]i following addition of oxytocin in the absence of extracellular calcium (greater than 15-min observation), indicating that this response requires addition of extracellular calcium. Furthermore, in the presence of extracellular calcium, oxytocin elicits only a single [Ca2+]i transient, with additional transients occurring only after 10 min and in more densely plated cells (data not shown).
Expression of Trp mRNAs in Human Myometrium Tissue and the PHM1-41 Cell Line
Reverse transcription-polymerase (RT-PCR) chain reaction was employed to explore the expression of specific Trp mRNAs in PHM1-41 cells and in pregnant human myometrium, using the primers listed in Table 1. Figure 2A shows that fragments of hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7 of the expected sizes were detected in mRNA from both PHM1-41 cells and in pregnant human myometrium. Lanes 1 and 7 show the product obtained with primers spanning the region specific to the hTrp1 variant, hTrp1. Using a primer that spanned an alternative spliced exon, we obtained 547- and 443-base pair (bp) Trp1 products corresponding with 100% sequence identity to hTrp1 and hTrp1ß splice variants, respectively (lanes 2 and 8). Lanes 3 and 9 contain the expected PCR fragments corresponding, with 99% identity, to hTrp3, and lanes 4 and 10 contain the expected products, with 99% identity, to hTrp4. Similarly, products with 99% identity to hTrp6 (lanes 5 and 11) and hTrp7 (lanes 6 and 12) were produced. In addition, using a primer that spanned an alternative spliced exon, we obtained 496- and 245-bp Trp4 products corresponding, with 100% sequence identity, to hTrp4 and hTrp4ß splice variants, respectively (Fig. 2B). In contrast, several attempts to isolate hTrp5 products by amplification of several different regions were unsuccessful. In addition, no products were observed for hTrp 2, consistent with the observation that Trp2 is a pseudogene in humans.
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Subsequently, we have determined the entire sequence of several Trp cDNAs derived from PHM1-41 mRNA. hTrp1, hTrp3, hTrp4, and hTrp6 from human myometrium all exhibited 100% identity to the sequences cited in Table 1.
Additional Trp Channel Splice Variants Expressed in PHM1-41 Cells
A number of Trp mRNA splice variants have been reported. In addition to some variants already described, we have identified several new splice variants in PHM1-41 mRNA. In addition to hTrp1 and hTrp1, we also isolated PCR products representing two hTrp1 splice variants not previously described (Fig. 3). These variants, which we designate hTrp11 and hTrp12, consist of 121- and 55-bp insertions at positions 654979–655099 and 657262–657316 in the genomic sequence (NT_005772.7, human chromosome 3 working draft), respectively. The sequences have 100% identity with genomic sequences at these positions and represent potential new exons, designated 9a and 9b, between exon 9 and exon 10. Examination of the intron/exon junctions revealed splice sites reasonably close to consensus and to those found between exons 9 and 10. The alignment of these splice variants in relation to the transmembrane domain region predicted from hydropathy analysis and by analogy to the analysis of Vannier et al. [29] is shown in Figure 3B. As previously described [15], the hTrp1 variant containing a 102-bp deletion corresponding to hTrp1 exon 3 results in an in-frame deletion in the N-terminal region of the molecule at the end of an ankyrin repeat (Fig. 3B). In contrast, the insertions of exons 9a and 9b do not maintain reading frame in the transcripts and would introduce stop codons just upstream of the fifth transmembrane region of the molecule.
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Alternatively spliced transcripts have not been reported for human Trp3. In addition to obtaining cDNA corresponding to the previously described hTrp3, we also isolated another form containing a 48-bp insertion between exon 8 and exon 9 (Fig. 3), which we designate hTrp3ß. The insert represents a potential new exon (8a) with 100% identity at positions 344494–344541 to the genomic sequence (NT_022755, human chromosome 4 working draft). Insertion of this sequence retains the open reading frame of the transcript. The intron/exon junctions for 8a were close to consensus and almost identical to those for exons 8 and 9. If translated, this splice variant would give rise to a product with a 16 amino acid addition just upstream from the calmodulin/IP3 receptor binding motif [30]. These new sequences have been assigned accession numbers AF483645 (hTrp11), AF483646 (hTrp12), and AF483647(hTrp3ß) in the NCBI GenBank.
Trp Channel Proteins Expressed in Pregnant Human Myometrium and PHM1-41 Cells
In Western blot analysis, an anti-Trp1 polyclonal antibody detected a protein with an approximate size of 95 kDa in both human myometrial and PHM1-41 membranes (Fig. 4A). The appearance of this band was blocked by preabsorption of the antibody with the immunizing peptide (data not shown). Using an anti-Trp3 polyclonal antibody, a 105-kDa band was detected in human myometrial and PHM1-41 membranes and in COS-M6 cells overexpressing full-length hTrp3 (Fig. 4B). A signal was detected at 110 kDa with anti-Trp4 antibody in human myometrium, PHM1-41 cells, and COS-M6 cells overexpressing kidney hTrp4 (Fig. 4C). A weak signal was detected at 130 kDa with an anti-Trp6 antibody in human myometrial membranes but not in PHM1-41 membranes (Fig. 4D). Bands were also detected at 65 kDa in human myometrium and 66 kDa in PHM1 cells, similar to what is reported in rat brain membrane by the manufacturer. These bands were not observed when the antibody was preabsorbed with immunizing peptide (data not shown). It is difficult to assess whether the weak signal for the putative intact protein is a reflection of the relative abundance of protein or the potency of the antibody. The lower molecular weight bands could represent proteolytic cleavage products, but this is yet to be demonstrated. A specific antibody against hTrp7 is currently not available.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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q/ll subfamily and subsequent activation of enzymes of the PLCß subfamily result in generation of inositol 1,4,5-trisphosphate that triggers release of Ca2+ from intracellular stores [3–5]. Our initial studies in rat myometrial cells demonstrated that the ability of oxytocin, norepinephrine, and carbachol to increase [Ca2+]i was partially dependent on extracellular Ca2+ and was not inhibited by the L-type Ca2+ channel blocker D600 [31]. In PHM1-41 cells derived from pregnant human myometrium, a similar dependence on extracellular Ca2+ and lack of effect of nifedipine was observed [11]. While voltage-operated Ca2+ channels clearly play a role in normal myometrium [7], these channels were not detectable in PHM1-41 cells by RT-PCR or response to depolarization (unpublished observations). In PHM1-41 cell suspensions, the oxytocin-stimulated increase in [Ca2+]i was inhibited almost entirely with phospholipase C inhibitors [11], consistent with contribution of a capacitative Ca2+ entry mechanism that involves one or more components of the G-protein signaling pathway. In addition, Ca2+ entry was enhanced by thapsigargin-mediated depletion of intracellular Ca2+ stores [11]. The data presented here confirm those results on a single-cell basis, with each cell serving as its own control for response to addition of extracellular Ca2+. The response to thapsigargin was relatively uniform between cells, as might be expected for response to a general inhibition of the endoplasmic reticulum Ca2+-ATPase presumably present in all cells. There was considerably more heterogeneity in the capacitative response to oxytocin, however. This may reflect some heterogeneity in the number and types of channel proteins expressed in individual cells. The PHM1-41 cells were derived from the initial immortalization mixture by subcloning but were not carried to limiting cell dilution because of their poor growth at low density, making this a reasonable explanation. Nonetheless, the data show that PHM1-41 myometrial cells exhibit stimulated Ca2+ entry activated both by G-protein coupled receptor-mediated pathways and by direct store depletion. Mechanisms governing capacitative Ca2+ entry are not completely understood. Theories to explain the signaling pathways include the generation of a small, diffusable message, a vesicle-mediated process, and conformational coupling mediated by interaction with proteins such as the IP3 receptor [3, 4]. The Drosophila Trp and TrpL proteins display some properties of capacitative Ca2+ channels, particularly when expressed together. Mammalian Trp proteins 1–7 also exhibit some properties of these channels [3–5]. However, when overexpressed alone, they are sometimes constitutively active and often nonselective with respect to ion preference. It has been postulated that the channel consists of tetramers, with the specific composition defining its properties in a given cell. Trp proteins range in their expression from being ubiquitous to tissue specific; expression patterns also differ between species [5]. Although Trp4, Trp6, and Trp7 mRNA have recently been reported to be expressed in a number of smooth muscles [32], to date, only hTrp4 mRNA has been reported in uterus [33]. In this article, we show that mRNA for hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7 mRNA is present in pregnant human myometrium and in PHM1-41 cells. Using several primer pairs, we were unable to obtain RT-PCR products corresponding to hTrp5, indicating that, if present at all, it is expressed in very low amounts. The fact that the same Trp mRNAs were found both in pregnant human myometrium and the cell line provides additional evidence that PHM1-41 cells have retained many of the characteristics of human myometrial tissue.
Trp pre-mRNAs apparently undergo multiple alternative splicing events. Two splice variants of hTrp1 mRNA have been reported [14, 15, 34]. Recently, alternative splice variants have also been reported for rat Trp3 [35], human and mouse Trp4 [27, 32], rat Trp6 [36], and mouse and canine Trp7 [32]. These variations include deletions of previously described exons in various regions, including in the N-terminus, in the membrane spanning region, and in the C-terminus. Although most of these variants have not been studied extensively, in a few cases, differences in phenotype have been noted. For example, rTrp3sv, lacking most of the homologous N-terminal hTrp3 sequence and ankyrin repeats, was not activated by GPCR or thapsigargin and had properties more similar to a Ca2+-activated cation channel [35]. The rTrp4ß and hTrp4ß, lacking part of the C-terminal region of a calmodulin/IP3R interaction site [37, 38], enhanced GPCR-stimulated Ca entry [27]. The longer rTrp was less effective and hTrp4, which may contain a putative autoinhibitory domain, was almost inactive. The functional importance of the expression of both of these forms in myometrium remains to be determined. The rTrp6A enhanced calcium entry stimulated by GPCR activation and diacylglyceride, whereas rTrp6B, lacking an N-terminal segment near the initiation codon, was only sensitive to GPCR activation and rTrp6C, lacking this segment and also a segment just downstream of the sixth transmembrane domain, was not active [36].
In myometrium, we have detected the reported variants of hTrp1 and two new splice variants representing insertions of two previously unidentified exons, 9a and 9b, in the C-terminal tail region of the molecule. The 121- and 55-bp inserts in hTrp11 and hTrp12, respectively, introduce stop codons in the putative protein just upstream of transmembrane domain 5. If translated, these truncated Trp proteins might disrupt capacitative Ca channel function if incorporated into a heterotetramer. Putative truncated forms of mTrp4 have been reported but not characterized. The relative importance of such truncated products remains to be determined.
We also report a new hTrp3 splice variant that represents an insertion of an exon between exons 8 and 9, adding 18 amino acids in the C-terminal region just upstream of the calmodulin/IP3 receptor interaction site. Because the C-terminal region of Trps has been implicated in interaction with scaffolding proteins as well, such an insertion could potentially influence these interactions or even create additional ones.
We were able to detect expression of hTrp1, hTrp3, and hTrp4 proteins in both human myometrial tissue and PHM1-41 cell membranes and hTrp6 protein in human myometrium. The sizes of these proteins were in the range of those reported by others [16, 33, 39]. The presence of multiple Trps raises the possibility that channels could be comprised of homo- and heterotetramers, each with unique properties, in this tissue. The myometrial cells respond to both GPCR stimulation and to thapsigargin with enhanced Ca2+ entry. To date, it has not been possible to correlate expression of specific channels with properties of capacitative Ca2+ entry in native cells. For example, in native HEK293 cells, Trp1, Trp2, Trp4, and Trp6 are expressed, but no diacylglycerol-sensitive Ca2+ entry, a property of Trp6, was noted [33]. A recent comparison notes similar properties of capacitative Ca2+ currents in several cell lines, even though the cells expressed different Trp isoforms [40]. On the other hand, antisense Trp4 significantly inhibited endogenous capacitative Ca2+ entry in murine L cells [13] and in SBAC cells expressing bTrp1 and bTrp4 [26].
It remains to be determined which of the Trp proteins expressed in human myometrium contribute to capacitative Ca2+ entry in a substantive way. In the meantime, these data add additional support to the hypothesis that GPCR stimulation leads to capacitative Ca2+ entry in addition to intracellular Ca2+ release in the myometrium. These two actions, in addition to entry through voltage-activated Ca2+ channels, contribute to the increase in contractile activity of the tissue. Because some Trp proteins are inhibited by Ca2+ or calmodulin [30, 41], the potential for Trp proteins in negative feedback on this response also exists.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH-HD09618, NIH-HD38970, and Lalor Foundation Fellowships (M.Y. and A.G.). 
2 Correspondence: Barbara M. Sanborn, Department of Biochemistry and Molecular Biology, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225. FAX: 713 500 0652; barbara.m.sanborn{at}uth.tmc.edu
3 Current address: Lexicon Genetics, 4000 Research Forest Dr., The Woodlands, TX 77381
Accepted: April 18, 2002.
Received: February 1, 2002.
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REFERENCES |
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