Construction of a Gal1-Regulated Yeast Cdna Expression Library and Its Application to the Identification of Genes Whose Overexpression Causes Lethality in Yeast

We have constructed a galactose-inducible expression library by cloning yeast cDNAs unidirectionally under control of the GAL1 promoter in a centromeric shuttle vector. Eleven independent libraries were made each with an average size of about 1 x 10(6) clones, about 50 times larger than the reported mRNA population in a yeast cell. From this library, LEU2 and HIS3 cDNAs were recovered at a frequency of about 1 in 10(4) and in 12 out of 13 cases these were expressed in a galactose-dependent manner. Sequence analysis of leu2 and his3 complementing cDNAs indicates that they contain all the coding sequence and much of the 5' untranslated region. To test the utility of the library for the identification of genes whose overexpression confers a specific phenotype, we screened 25,000 yeast transformants for lethality on galactose. Among 15 clones that showed galactose inducible lethality were cDNAs encoding structural proteins, including ACT1 (actin), TUB2 (beta-tubulin) and ABP1 (actin-binding protein 1), and genes in signal transduction pathways, including TPK1 (a cAMP-dependent protein kinase) and GLC7 (type 1 protein phosphatase). cDNAs overexpressing NHPB (nonhistone protein B) and NSR1 (nuclear sequence recognition protein) were also found to be lethal. Among these, ACT1 was isolated four times, and NSR1 three times. The useful features of this library for cDNA cloning in yeast by complementation, and for the identification of genes whose over-expression confers specific phenotypes, are discussed.     

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.     

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.     

Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence.

The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized. Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E. coli. In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity. The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2. Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays. Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase. Here we examine the substrate sequence specificity of Pim-1 kinase in detail. Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly). The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition. Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above. Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X', where X' is likely neither a basic nor a large hydrophobic residue.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.     

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Tomato protein kinase 1b mediates signaling of plant responses to necrotrophic fungi and insect herbivory

The tomato protein kinase 1 (TPK1b) gene encodes a receptor-like cytoplasmic kinase localized to the plasma membrane. Pathogen infection, mechanical wounding, and oxidative stress induce expression of TPK1b, and reducing TPK1b gene expression through RNA interference (RNAi) increases tomato susceptibility to the necrotrophic fungus Botrytis cinerea and to feeding by larvae of tobacco hornworm (Manduca sexta) but not to the bacterial pathogen Pseudomonas syringae. TPK1b RNAi seedlings are also impaired in ethylene (ET) responses. Notably, susceptibility to Botrytis and insect feeding is correlated with reduced expression of the proteinase inhibitor II gene in response to Botrytis and 1-aminocyclopropane-1-carboxylic acid, the natural precursor of ET, but wild-type expression in response to mechanical wounding and methyl-jasmonate. TPK1b functions independent of JA biosynthesis and response genes required for resistance to Botrytis. TPK1b is a functional kinase with autophosphorylation and Myelin Basis Protein phosphorylation activities. Three residues in the activation segment play a critical role in the kinase activity and in vivo signaling function of TPK1b. In sum, our findings establish a signaling role for TPK1b in an ET-mediated shared defense mechanism for resistance to necrotrophic fungi and herbivorous insects.     

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.     

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.     

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.     

Modulating protein folding rates in vivo and in vitro by side-chain interactions between the parallel beta strands of green fluorescent protein

We have identified pairs of residues across the two parallel beta strands of green fluorescent protein that facilitate native strand register of the surface-exposed beta barrel. After constructing a suitable host environment around two guest residues, minimizing interactions of the guest residues with surrounding side-chains yet maintaining the wild-type protein structure and the chromophore environment, we introduced a library of cross-strand pairings by cassette mutagenesis. Colonies of Escherichia coli transformed with the library differ in intracellular fluorescence. Most of the fluorescent pairs have predominantly charged and polar guest site residues. The magnitude and the rate of fluorescence acquisition in vivo from transformed E. coli cells varies among the mutants despite comparable levels of protein expression. Spectroscopic measurements of purified mutants show that the native protein structure is maintained. Kinetic studies using purified protein with fully matured chromophores demonstrate that the mutants span a 10-fold range in folding rates with undetectable differences in unfolding rates. Thus, green fluorescent protein provides an ideal system for monitoring determinants of in vivo protein folding. Cross-strand pairings affect both protein stability and folding kinetics by favoring the formation of native strand register preferentially to non-native strand alignments.     

Recombinant rabbit muscle casein kinase I alpha is inhibited by heparin and activated by polylysine.

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.     

A system for dual protein expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Maize Glutamine Synthetase Cdnas: Isolation by Direct Genetic Selection in Escherichia Coli

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II by expression of mutated cDNAs in Escherichia coli.

The cDNAs encoding the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were ligated into the bacterial expression vector pET and expressed in Escherichia coli. The bacterially expressed alpha and beta subunits exhibited Ca2+/calmodulin-dependent activity and were easily purified to apparent homogeneity from cell extracts. To determine the minimum size required for catalytic activity and the properties of the calmodulin-binding domain, mutated CaM kinase II cDNAs were expressed in E. coli and the enzymatic property of expressed proteins was examined. The replacement of Thr-286 of the alpha subunit with the negatively charged amino acid Asp or that of Arg-283 with the neutral amino acid Gly induced the partially Ca2+ independent activity. The mutant enzymes alpha-I(delta 283-478) and alpha-II(delta 359-478), which truncated the C-terminal region of the alpha subunit, exhibited CaM kinase II activity and the activities of alpha-I(delta 283-478) and alpha-II(delta 359-478) were completely independent of and partially dependent on Ca2+ and calmodulin, respectively. However, the truncated protein alpha(delta 250-478), which was only 33 amino acids shorter than the alpha-I(delta 283-478) protein had no enzymatic activity, indicating that alpha-I(delta 283-478) was close to the minimum size of the active form. The mutant enzyme alpha(delta 291-315), which lacked the calmodulin-binding domain exhibited Ca2+ independent activity. The molecular mass was, however, smaller than that expected from the amino acid sequence. The mutant enzyme alpha(delta 304-315), which lacked the C-terminal half of the calmodulin-binding domain of the alpha subunit, however, exhibited Ca(2+)-independent activity without a reduction in molecular size, indicating that residues 304-315 of the alpha subunit constituted the core calmodulin-binding domain.