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First published online August 13, 2004; 10.1104/pp.104.041442 Plant Physiology 135:2150-2161 (2004) © 2004 American Society of Plant Biologists Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor1Division of Applied Life Science (BK21 Program), Environmental Biotechnology Research Center and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660–701, Korea
The Ca2+-binding protein calmodulin mediates cellular Ca2+ signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions –858 and –728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.
Plant cells, like animal cells, elevate their cytosolic free-calcium levels ([Ca2+]cyt) with varying amplitude, frequency, and duration in response to a variety of external stimuli (Thomas et al., 1996  ; Berridge, 1997; McAinsh and Hetherington, 1998). The stimulus-specific [Ca2+]cyt transients are sensed by intracellular Ca2+-binding proteins, of which calmodulin (CaM) is one of the best characterized (Chin and Means, 2000; Snedden and Fromm, 2001). CaM is a ubiquitous intracellular mediator of Ca2+ signals having four helix-loop-helix Ca2+-binding motifs referred to as EF-hands (Babu et al., 1988). The Ca2+-bound CaM transduces the signals into many cellular processes through modulation of a variety of CaM-binding proteins, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (Liao et al., 1996; Snedden and Fromm, 1998; Lee et al., 1999a; Zuhlke et al., 1999).
In plant cells, in contrast to mammalian cells, multiple CaM genes code for a number of CaM isoforms. This has been shown in wheat (Triticum aestivum; Yang et al., 1996
SCaM-4 can be distinguished from SCaM-1 by the target enzymes that it can activate (Lee et al., 1995a
All SCaM isoforms, including SCaM-4, are ubiquitously expressed in various plant tissues and show similar subcellular localization patterns to those of SCaM-1 (Lee et al., 1995a Although we know that the expression of CaM isoforms is differentially regulated by specific external stimuli, the cis- and trans-acting elements involved in plant CaM gene expression have not been well characterized. In this study, we have isolated and characterized the promoter sequence of the SCaM-4 gene. Core cis-acting elements that regulate expression of the SCaM-4 gene in response to pathogen infection or salt stress were identified within the SCaM-4 promoter between –1,215 and –1,150 bp, and between –858 and –728 bp. Here we report that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression.
Isolation of the SCaM-4 Promoter and Analysis of Tissue-Specific Expression of the ScaM-4 Promoter-  -Glucuronidase Reporter GeneTo characterize the regulatory mechanisms controlling transcription of the SCaM-4 gene, we isolated its promoter region. Figure 1 shows the sequence of the SCaM-4 promoter (–1,286 bp to +765 bp), which extends into the 5'-untranslated region (GenBank accession no. AY052528). For comparative purposes, 2.4 kb of the 5'-upstream region of SCaM-1 was isolated from a soybean genomic library using SCaM-1 cDNA as a probe (GenBank accession no. AY052527; data not shown). We used a primer extension analysis to map the start site of SCaM-4 transcription. Two long extension products were detected 689 bp and 683 bp upstream of the first ATG site, suggesting heterogeneity in the mRNA 5' ends or premature arrest of the reverse transcriptase (data not shown). The G residue corresponding to the longer extension product was taken to be the transcription start site and was numbered +1. As shown in Figure 1, a putative TATA box sequence is located upstream (nucleotides –33 to –37) of the transcription start site.
We then examined the tissue-specific expression pattern of an ScaM-4 promoter- -glucuronidase (GUS) reporter in transgenic Arabidopsis to see whether it matched the expression pattern of ScaM-4 gene in soybean (Lee et al., 1995a, 1999b). Figure 2 shows representative examples of the tissue-specific expression of the SCaM-4 promoter-GUS gene. The SCaM-4 promoter-GUS was expressed primarily in the apical meristem (Fig. 2D) and hypocotyl regions of transgenic Arabidopsis seedlings (Fig. 2, C and F). The GUS staining patterns in transgenic Arabidopsis seedlings were similar to the expression patterns of SCaM-4 mRNA and SCaM-4 protein in soybean seedlings (Lee et al., 1995a, 1999b).
Analysis of the Effect of Signaling Molecules on Expression of the SCaM-4 Promoter-GUS Reporter Gene
Expression of plant CaM genes has been shown to respond to various environmental stresses including light, phytohormones, touch, wounding, high salinity, and pathogens (Jena et al., 1989
We then examined the effects of these treatments on GUS reporter gene expression in Arabidopsis leaf protoplasts, a plant transient expression system. After treatment with various biotic and abiotic signals, we determined the level of induction of GUS activity with reference to luciferase (LUC) activity. The GUS activity of the SCaM-4 promoter-GUS reporter was enhanced about 3- to 7-fold when treated with PsD, glycol chitin, NaCl, or Ca2+-ionophore A23187 (Fig. 3D). However, the other treatments did not increase GUS activity. We also examined the effects of the treatments on the expression of 2.4-kb SCaM-1 promoter-GUS construct. No GUS induction was observed for the SCaM-1 promoter in Arabidopsis protoplast cultures, similar to the expression pattern found in soybean seedlings (Fig. 3E). Overall, these experiments show that Arabidopsis can be a useful system in which to study the pathogen and NaCl responsive regulatory elements of the ScaM-4 promoter.
To determine the specific regions of the promoter that are involved in SCaM-4 induction by pathogen or NaCl treatments, a series of 5' deletions were made in the SCaM-4 promoter region (Fig. 4A). Each construct was transiently introduced into Arabidopsis protoplasts by polyethylene glycol-mediated transformation, and GUS activity was assayed after treatment with 150 mM NaCl, or PsD for 12 h. The GUS reporter gene was strongly induced by pathogen or NaCl in constructs containing deletions up to –1,286 (pBI 4D1) or –858 (pBI 4D2), but this induction was completely lost in the construct containing a deletion up to –566 (pBI 4D3). Furthermore, the pBI 4delA construct, containing nucleotides –1,286 to –728, showed maximal GUS induction after treatment with pathogen or NaCl (about 14- and 19-fold, respectively), a pattern of induction very similar to that of the SCaM-4 2-kb promoter (Fig. 4B).
To determine the region(s) within the –1,286 to –728 bp region that are responsible for induction by pathogen and NaCl treatments, the SCaM-4 promoter region was further divided into six overlapping fragments of 100 to 200 bp in length, and the fragments were fused to the upstream region of the TATA minimal promoter contained within the pDel. 151-8 vector (Sundaresan et al., 1995
From GUS assays in Arabidopsis protoplasts containing the deletion constructs of the SCaM-4 promoter in vivo, we identified nucleotides –1,286 to –1,065 and –858 to –728 as important elements for pathogen and NaCl responses. To test whether these regions interact specifically with nuclear proteins, we divided the 1.3-kb promoter region into five fragments (fragments A–E), as shown in Figure 5A. Each of the five double-stranded fragments was used in an initial series of electrophoretic mobility shift assays (EMSAs) with soybean nuclear extracts from W82 cells treated for 1 h with 10 mM MgCl2 (control), pathogen (Psg), or NaCl. Fragments containing the –1,286 to –1,065 (A) and –858 to –549 (C) regions each gave one major retarded band when incubated with Psg- or NaCl-treated nuclear extracts (Fig. 5A). Fragment C (–858 to –549) was further subdivided into three overlapping fragments, C-1, C-2, and C-3, and each was used in EMSAs with the nuclear extracts described above. Only the C-1 region fragment (–858 to –728) showed a strong mobility shift when incubated with Psg-treated nuclear extracts (Fig. 5B). The mobility shift was completely blocked by the addition of a 50-fold molar excess of unlabeled C-1 but not by an excess of unlabeled C-2 or C-3. EMSAs using the C-1 region as a probe with heat-treated (65°C, 5 min) or proteinase K-treated nuclear extracts, showed that the DNA-binding complex of the C-1 region was heat stable but sensitive to proteinase K digestion (data not shown).
To more precisely define the position of the protein binding site, we designed nine double-stranded oligonucleotides of 15 to 30 bp in length (E1–E9, Fig. 5C), which were used as C-1 competitors in EMSAs. The E4 oligonucleotide completely blocked nuclear protein binding to the C-1 fragment (Fig. 5D). Examination of the sequence of the E4 fragment revealed that it contains a GT-1 cis-element (GAAAAA). DNase I footprinting assays using the C-1 fragment and pathogen-treated nuclear extracts confirmed that the GT-1 element is indeed recognized by nuclear factors (data not shown). The involvement of the GT-1 cis-element in binding to pathogen-treated nuclear extracts was tested using a subset of oligonucleotides derived from E4 fragments (Fig. 5E). While E4-1 (TAAGAAAAATAA) effectively bound to pathogen treated nuclear extracts, the mutations E4-1(M1; TAACAAAAATAA) and E4-1(M2; TAACCAAAATAA) caused significant reductions in protein binding (Fig. 5E). To examine whether the GT-1 cis-element of the SCaM-4 promoter, identified by in vitro DNA binding, actually plays a role in the cellular responses to pathogen and NaCl-induction, we generated a mutant ScaM-4 promoter (–1,286 to –728)-GUS construct that contains a GA to CC mutation in the GT-1 element (Fig. 6A). The pBI 4delA (–1,286 to –728) showed a 7- to 8-fold induction of the GUS reporter gene after treatment with NaCl or pathogen. However, the pBI 4delA M2 mutant construct repeatedly showed 4- to 5-fold induction by the same treatment, approximately a 30% reduction compared to the wild type promoter (Fig. 6B). This result shows that while the GT-1 element is involved in the expression of ScaM-4, the –1,065 to –1,286 region also plays a role in the NaCl- and pathogen-induced expression of the SCaM-4 gene (Fig. 5A).
Isolation of a Transcription Factor Interacting with the SCaM-4 GT-1 cis-Element
As a first approach to isolate the transcription factor that interacts with the GT-1 cis-element within the SCaM-4 promoter, we searched the complete genome of Arabidopsis. Seventeen sequences encoding trihelix DNA-binding factors (or GT transcription factors) have been found in the Arabidopsis genome (Ayadi et al., 2004
The full-length cDNA clones for the three AtGT-1-related proteins (AtGT-1, AtGT-4, and AtGT-3b) were isolated by the reverse transcription (RT)-PCR method. The expression patterns of the three AtGT-1-related genes were examined in pathogen- and NaCl-treated Arabidopsis plants by northern-blot analysis. As shown in Figure 7B, treatment of the plants with pathogen or NaCl resulted in a rapid increase in the transcription of AtGT-3b. However, the expression levels of the two other AtGT-1-related transcription factors were not changed by treatment with pathogen or NaCl. Therefore, AtGT-3b was selected as a good candidate for a transcription factor which binds to the GT-1 cis-element of the SCaM-4 promoter during the plant response to pathogen attack and NaCl stress. The AtGT-3b gene has an open reading frame of 870 bp, which would encode a protein of 289 amino acids with a molecular mass of approximately 31.8 kD (Fig. 7A). The deduced amino acid sequence of AtGT-3b contains two different nuclear localization signal sequences, one of which corresponds to a bipartite-type nuclear localization signal within a trihelix domain (KRNKLLWEVISNKMRDK) located between amino acids 65 and 81. The other corresponds to a simian virus 40 (SV 40)-type nuclear localization signal located in the C-terminal region (KKRK) encompassing amino acids 185 to 188. The AtGT-3b protein was further analyzed for interactions with the GT-1 cis-element in the SCaM-4 promoter in vitro and in a yeast selection system. To test the binding activity of AtGT-3b to the GT-1 cis-element within the E4 fragment of the SCaM-4 promoter, we produced a recombinant AtGT-3b protein fused to glutathione S-transferase (GST) in Escherichia coli. As shown in Figure 8A, the ability of the recombinant GST-AtGT-3b fusion protein to bind to the E4 oligonucleotide was validated by EMSA. The DNA binding specificity of GST-AtGT-3b was also confirmed by competition experiments (Fig. 8A). A 200-fold molar excess of unlabeled E4 oligonucleotide completely blocked E4 binding to GST-AtGT-3b (Fig. 8A, lane 9). In contrast, neither AtGT-1 nor AtGT-4 formed protein-DNA complexes under these conditions (data not shown).
The interaction of the AtGT-3b protein with the GT-1 cis-element was reconfirmed with a yeast selection system. We constructed a YM4271 yeast strain carrying integrated copies of HIS3 and lacZ as dual reporter genes with four tandem repeats of the E4 fragment upstream of the TATA element. The yeast cells were then transformed with AtGT-3b cDNA fused to the transcriptional activation domain of yeast GAL4 (Fig. 8B). As predicted by the in vitro binding assay, the AtGT-3b protein and the E4 fragment conferred HIS selection in the presence of 45 mM 3-AT, a competitive inhibitor of the HIS3 gene product (HIS3p). This result provides strong evidence for an interaction in the yeast system. In contrast, yeast cells carrying plasmids with cDNA inserts of AtGT-1 or AtGT-4 did not grow on medium lacking His in the presence of 45 mM 3-AT (data not shown).
The expression of plant CaM and CaM-like genes from a number of species is differentially regulated in response to external stimuli of both abiotic (e.g. light, gravity, heat, touch, cold, salinity, and drought) and biotic (e.g. phytohormones and pathogens) origins (Snedden and Fromm, 1998 , 2001). We have previously shown that SCaM-4 and SCaM-5 are more rapidly induced by fungal elicitors or pathogens than are the SCaM-1, -2, and -3 isoforms (Heo et al., 1999). Here we have shown that the mRNA of SCaM-4 is also significantly induced in response to salt stress (Fig. 3A). Furthermore, our group has already shown that SCaM-4 activates a different pattern of CaM-dependent enzymes than SCaM-1 (Lee et al., 2000). These findings suggest that particular Ca2+/CaM signaling pathways can be mediated by different CaM isoforms, which, in turn, may give plant cells the ability to have diverse cellular responses to Ca2+ signals. To elucidate the differences between SCaM-1 and SCaM-4 at the level of transcriptional regulation, we isolated the 5'-flanking regions of the SCaM-1 gene (2.4 kb) and the SCaM-4 gene (2 kb).
To further understand the upstream signaling mechanisms of SCaM-4 gene expression in response to pathogen and NaCl signals, we analyzed the cis- and trans-acting elements involved in SCaM-4 gene expression. Based on a report that Arabidopsis also contains SCaM-4/-5 gene homologs (Zielinski, 2002 Using EMSAs, we examined protein-DNA interactions with the SCaM-4 promoter and the soybean nuclear extracts and precisely identified the cis-acting elements involved in plant responses to pathogen attack and NaCl stress. Two promoter regions, –1,286 to –1,065 (A) and –858 to –566 (C), were critical for the SCaM-4 promoter binding of nuclear extracts prepared from pathogen- or NaCl-treated soybean suspension culture cells (W82). This result is in good agreement with the data obtained from the in vivo transient expression assay using Arabidopsis protoplasts. From a DNase I footprinting analysis and EMSA using synthetic oligonucleotides, we identified a GT-1 cis-element within a subfragment of the C region, the E4 fragment, as an important element involved in SCaM-4 gene expression (Figs. 5 and 6). Additionally, a base substitution analysis demonstrated that GA in the GT-1 cis-element (5'-GAAAAA-3') is required for binding to nuclear factor(s) in response to pathogen- or salt-induced stress. Together, these data imply that a GT-1-related transcription factor positively regulates SCaM-4 gene expression under the conditions of pathogen attack or NaCl stress.
The GT-1 cis-element, one of many cis-acting DNA elements found in plants, was first identified in pea (Pisum sativum) as the Box II element (5'GTGTGGTTAATATG3') in the promoter of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene (RBCS-3A; Green et al., 1987
In vitro experiments have shown that the GT-1 transcription factor can interact with the TFIIA-TBP-TATA complex, suggesting that GT-1 may activate transcription through direct interactions with the minimal preinitiation complex (Le Gourrierec et al., 1999 The data obtained from this study lead to a model in which environmental stresses induce SCaM-4 gene expression by mediating the binding of a GT-1-like transcription factor to the GT-1 cis-element (GAAAAA) within the –858 to –728 region of the SCaM-4 promoter. Additional binding event(s), mediated by yet to be defined trans-acting factor(s), may be required at the upstream –1,215 to –1,065 bp cis-element. Currently, we are characterizing the –1,286 to –1,065 bp region with respect to its contribution to the induction of SCaM-4 gene transcription in response to pathogens and NaCl. Interestingly, we have found a 65 bp sequence within the –1,215 to –1,150 region that is retarded in EMSAs from pathogen- or NaCl-treated nuclear extracts (data not shown). Further investigation into the regulation of SCaM-4 will involve characterization of other cis-elements and their cognate transcription factors. This will provide a better understanding of the roles played by DNA-protein interactions in SCaM-4 gene expression during plant defense responses.
Plant Materials and Bacterial and Yeast Strains
Soybean (Glycine max) cells (W82) were grown in suspension culture in Murashige and Skoog medium supplemented with 0.75 mg L–1 benzyl adenine, maintained at 25°C in the dark, and stirred at 130 rpm. Arabidopsis (ecotype Columbia) plants were used for the preparation of transgenic plants. For DNA cloning, Escherichia coli XL1-Blue MRF' and DH 5
The 5' upstream region of the SCaM-4 gene was obtained using the Universal Genome Walker kit (CLONTECH, Palo Alto, CA). First, separate aliquots of soybean genomic DNA were digested with five blunt-end restriction enzymes (EcoRV, ScaI, DraI, PvuII, and StuI), and ligated to Genome Walker adaptors. Primary PCR was performed using adaptor primer 1 (AP 1) and a SCaM-4 cDNA specific primer (5'-GTCCTCGGTAAGAAACAGACTCATCC-3'). The second PCR was performed using adaptor primer 2 (AP 2) and the same SCaM-4 cDNA specific primer. The amplified PCR products were examined on an agarose gel, and subcloned into the pGEM T-Easy vector. After sequencing of overlapping deletion products using the Erase-A-Base kit (Promega, Madison, WI), the 5' upstream region of the 2-kb SCaM-4 gene was connected by asymmetric PCR.
The upstream region of the SCaM-1 gene was isolated by screening a soybean (Glycine max cv Williams 82) genomic DNA library constructed in bacteriophage
Various tissues of transgenic Arabidopsis plants and W82 cells collected on filter papers (Whatman, Clifton, NJ) by vacuum filtration were used for isolation of total RNA as described (Park et al., 2002
For promoter analysis in transgenic plants and Arabidopsis protoplasts, SCaM-4 promoter-GUS-NOS cassette constructs were used. Deleted promoters were cloned into the SalI/BamHI sites of the binary vector pBI 101 (CLONTECH). The following deletion derivatives were cloned into the SalI/BamHI site of the binary vector, pBI 101: a SCaM-1 promoter containing a fragment from –2,230 to +84, named pBI 1D1, and a SCaM-4 promoter containing various fragments (–1,286 to +750, pBI 4D1; –858 to +750, pBI 4D2; –566 to +750, pBI 4D3; –217 to +750, pBI 4D4; and +34 to +750, pBI 4D5). For characterization of the promoter in more detail, the –1,286 to –728 bp region of the SCaM-4 promoter was subdivided into six different fragments and ligated into the region upstream of the TATA-containing minimal promoter of the pDel. 151-8 vector (Sundaresan et al., 1995
Isolation of Arabidopsis protoplasts and polyethylene glycol-mediated DNA transfection were performed as described previously (Abel and Theologis, 1994
To generate transgenic Arabidopsis plants (ecotype Columbia), pBI 4D1 and pBI 101 plasmids were introduced into Agrobacterium tumefaciens GV3101 by electroporation, and Arabidopsis plants were transformed by vacuum infiltration (Clough and Bent, 1998
Different pathogenic bacteria (108 cfu/mL) were used for infection of the two plant species. Pseudomonas syringae pv glycinea carrying avrA (Psg) was used for infection of soybean suspension culture cells (W82), and P. syringae pv tomato DC3000 (PsD) was used for Arabidopsis plants. Bacteria grown in liquid King's medium were washed and resuspended in 10 mM MgCl2 (King et al., 1954
Nuclear extracts were prepared from W82 cells that had been treated with MgCl2 (mock inoculation), pathogen, or 150 mM NaCl for about 1 h using a procedure described previously (Nagao et al., 1993
Total RNA was extracted from the pathogen- or 150 mM NaCl-treated samples of 4-week-old Arabidopsis seedlings. Total RNA (5 µg) was reverse-transcribed in a 50-µL reaction volume with 10 ng of oligo(dT)17 primer using Superscript RTase according to the manufacturer's protocols (BRL Life Technologies, Grand Island, NY). The following oligonucleotides were synthesized for amplification of GT-1–related cDNAs in Arabidopsis (Ayadi et al., 2004
An AtGT-3b cDNA fragment was prepared by PCR and cloned into the BamHI and XhoI sites of the pGEX-2T-linker I vector (Amersham Pharmacia Biotech, Uppsala). Using E. coli strain BL21 (pLys S) DE3, GST::AtGT-3b was overexpressed, and the bacterial supernatant was used for gel mobility shift assays. For analysis in the yeast selection system, construction of reporter plasmids and selection of the yeast reporter strain were performed according to the manufacturer's protocol (CLONTECH). To generate the AD-fused AtGT-3b cDNA construct, a BamHI/PstI fragment of AtGT-3b cDNA was ligated into the pGAD424 vector. Positive interactions were verified by judging yeast growth on SD medium containing 45 mM 3-AT and assaying for Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY052528 for SCaM-1 promoter and AY052527 for SCaM-4 promoter.
We thank Dr. C. Lamb for providing the soybean suspension-culture cells, W82. Received February 22, 2004; returned for revision May 20, 2004; accepted May 24, 2004.
1 This work was supported by a Basic Research Grant (grant no. R02–2002–000–00009–0), by the National Research Laboratory program (2000–N–NL–01–C–236), by the Crop Functional Genomics Center of the 21st Century Frontier Research Program CG1512, by the Gyeongnam High Tech Foundation (2001), and by the Ministry of Agriculture and Forestry (grant no. 298049–4 to M.J.C.), and partially by the Environmental Biotechnology Research Center (grant no. R15–2003–012–02003–0), by the Crop Functional Genomics Center of the 21st Century Frontier Research Program CG1124, and by the Center for Plant Molecular and Genetic Breeding Research, KOSEF in Korea (grant to J.C.H.). Y.H.K., C.Y.P., and B.C.M. were supported by scholarships from the BK21 program, Ministry of Education and Human Resources Development in Korea. 
2 Present address: Department of Biology, Coker Hall, Room 108, CB 3280, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041442. * Corresponding authors; e-mail choslab{at}nongae.gsnu.ac.kr; fax 82–55–759–9363.
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ABSTRACT
RESULTS
(Stratagene, La Jolla, CA) were used as bacterial strains. The expression of the GST-fusion protein was performed in E. coli, BL21 (pLys S) DE3. The yeast strain YM4271 (MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-901, tyr1-501, gal4-
512, gal80-
Fix II (Stratagene, Heidelberg, Germany). A 2.4-kb internal EcoRI fragment that hybridized to the SCaM-1 cDNA probe was subcloned into the multiple cloning site of the pBluescript II SK (–) vector (Stratagene, La Jolla, CA). A sequential series of overlapping deletions from both ends were made using the Erase-A-Base kit (Promega) according to the manufacturer's protocol and sequenced. 
