Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution.

Pin1 is a phosphorylation-dependent member of the parvulin family of peptidyl-prolyl isomerases exhibiting functional conservation between yeast and man. To perform an unbiased analysis of the regions of Pin1 essential for its functions, we generated libraries of randomly mutated forms of the human Pin1 cDNA and identified functional Pin1 alleles by their ability to complement the Pin1 homolog Ess1 in Saccharomyces cerevisiae. We isolated an extensive collection of functional mutant Pin1 clones harboring a total of 356 amino acid substitutions. Surprisingly, many residues previously thought to be critical in Pin1 were found to be altered in this collection of functional mutants. In fact, only 17 residues were completely conserved in these mutants and in Pin1 sequences from other eukaryotic organisms, with only two of these conserved residues located within the WW domain of Pin1. Examination of invariant residues provided new insights regarding a phosphate-binding loop that distinguishes a phosphorylation-dependent peptidyl-prolyl isomerase such as Pin1 from other parvulins. In addition, these studies led to an investigation of residues involved in catalysis including C113 that was previously implicated as the catalytic nucleophile. We demonstrate that substitution of C113 with D does not compromise Pin1 function in vivo nor does this substitution abolish catalytic activity in purified recombinant Pin1. These findings are consistent with the prospect that the function of residue 113 may not be that of a nucleophile, thus raising questions about the model of nucleophilic catalysis. Accordingly, an alternative catalytic mechanism for Pin1 is postulated.

The yeast peptidyl proline isomerases FPR3 and FPR4, in high copy numbers, suppress defects resulting from the absence of the E3 ubiquitin ligase TOM1.

Tom1p is a 3268-amino acid protein with extensive homology to the hect-domain class of E3 ubiquitin ligases. Disruption of the TOMI gene results in temperature sensitivity for growth. Genes encoding the peptidyl proline isomerases Fpr3p and Fpr4p, when present on multicopy plasmids, will suppress this temperature-sensitive growth phenotype. FPR3 can also suppress the mating defect seen in tom1 strains. Suppression is specific for disruption of TOM1, since FPR3 does not restore wild-type growth to strains lacking the E2 ubiquitin-conjugating enzyme Rad6p or the transcriptional regulator Ngglp. Interestingly, the peptidyl proline isomerase domains of Fpr3p and Fpr4p are not required for suppression; rather the essential sequences include about 170 highly conserved residues at the proteins' N-termini. Previously we found that Tomlp plays a role in gene regulation. Since overexpression of FPR4 does not suppress the reduced expression of the ARG1 promoter found in tom1 deletion strains, Tom1p probably has one or more functions beyond its involvement in gene expression.

NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p.

Post-translational acetylation of histone H4 N-terminal tail in chromatin has been associated with several nuclear processes including transcription. We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4. NuA4 has an apparent molecular mass of 1.3 MDa. All four conserved lysines of histone H4 can be acetylated by NuA4. We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast. Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature-sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature. Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM-related essential gene homologous to human TRRAP, an essential cofactor for c-Myc- and E2F-mediated oncogenic transformation. Finally, the ability of NuA4 to stimulate GAL4-VP16-driven transcription from chromatin templates in vitro is also lost in the temperature-sensitive esa1 mutant. The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator-interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control.

Tra1p is a component of the yeast Ada.Spt transcriptional regulatory complexes.

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.

TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes.

The hect-domain has been characterized as a conserved feature of a group of E3 ubiquitin ligases. Here we show that the yeast hect-domain protein TOM1p regulates transcriptional activation through effects on the ADA transcriptional coactivator proteins. Null mutations of tom1 result in similar defects in transcription from ADH2 and HIS3 promoters, and enhanced transcription from the GAL10 promoter as do null mutations in ngg1/ada3. Strains with disruptions of both ngg1 and tom1 have the same phenotype as strains with a disruption of only ngg1 implying that these genes are acting through the same pathway. In the absence of TOM1p, the normal associations of the ADA proteins with SPT3p and the TATA-binding protein are reduced. The action of TOM1p is most likely mediated through ubiquitination since mutation of Cys3235 to Ala, corresponding residues of which are required for thioester bond formation with ubiquitin in other hect-domain proteins, results in similar changes in transcription as the null mutation. A direct role for TOM1p in regulation of ADA-associated proteins is further supported by the finding that SPT7p is ubiquitinated in a TOM1p-dependent fashion and that TOM1p coimmunoprecipitates with the ADA proteins.

Identification of native complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2.

NGG1p/ADA3p and ADA2p are dual function regulators that stimulate or inhibit a set of yeast transcriptional activator proteins. In vitro, NGG1p and ADA2p associate in a complex that also contains GCN5p (Horiuchi, J., Silverman, N., Marcus, G. A., and Guarente, L. (1995) Mol. Cell. Biol. 15, 1203-1209). We have found that NGG1p and ADA2p are coimmunoprecipitated from yeast whole cell extracts. In fact, <2% of cellular ADA2p was not associated with NGG1p. Also in agreement with their association in vivo, the stability of ADA2p and NGG1p depended on the presence of each other. In addition, three NGG1p- and ADA2p-containing peak fractions were resolved by Q-Sepharose Fast Flow ion-exchange chromatography of whole cell extract. The presence of another high molecular mass complex was supported by the separation of one of the NGG1p- and ADA2p-containing peak fractions by gel-filtration chromatography. Together, the combination of ion-exchange and gel-filtration chromatography suggests a total of four complexes, two with sizes of >2 MDa and single complexes of approximately 900 and 200 kDa. At least one of these complexes was found to associate with the TATA-binding protein (TBP) since TBP was present in immunoprecipitates with NGG1p. The association of TBP with the ADA proteins required amino acids 274-307 of NGG1p, a region of NGG1p required for activity. This supports a role for NGG1p in the interaction with TBP and suggests that the interaction with TBP is functionally relevant.

Transcriptional activation by yeast PDR1p is inhibited by its association with NGG1p/ADA3p.

NGG1p/ADA3p forms a coactivator/repressor complex (ADA complex) in association with at least two other yeast proteins, ADA2p and GCN5p, that is involved in regulating transcriptional activator proteins including GAL4p and GCN4p. Using a two-hybrid analysis, we found that the carboxyl-terminal transcriptional activation domain of PDR1p, the primary regulatory protein involved in yeast pleiotropic drug resistance, interacts with the amino-terminal 373 amino acids of NGG1p (NGG1p1-373). This interaction was confirmed by coimmunoprecipitation of epitope-tagged derivatives of NGG1p and PDR1p from crude extracts. An overlapping region of the related transcriptional activator PDR3p was also found to interact with NGG1p. Amino acids 274-307 of NGG1p were required for interaction with PDR1p. This same region is required for inhibition of transcriptional activation by GAL4p. The association between NGG1p1-373 and PDR1p may be indirect, possibly mediated by the ADA complex since the two-hybrid interaction required the presence of full-length NGG1. A partial requirement for ADA2 was also found. This suggests that an additional component of the ADA complex, regulated by ADA2p, may mediate the interaction. Transcriptional activation by a GAL4p DNA binding domain fusion of PDR1p was enhanced in ngg1 and ada2 disruption strains. Similar to its action on GAL4p, the ADA complex acts to inhibit the activation domain of PDR1p.

Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression.

NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p-NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p-independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373).

Defining the sequence specificity of the Saccharomyces cerevisiae DNA binding protein REB1p by selecting binding sites from random-sequence oligonucleotides.

We have used a random selection protocol to define the consensus and range of binding sites for the Saccharomyces cerevisiae REB1 protein. Thirty-five elements were sequenced which bound specifically to a GST-REB1p fusion protein coupled to glutathione-Sepharose under conditions in which more than 99.9% of the random sequences were not retained. Twenty-two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide-use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bind in vitro and activate transcription in vivo; however, the presence of non-conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low-affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within 'TATA-less' promoters.

GCN4p activation of the yeast TRP3 gene is enhanced by ABF1p and uses a suboptimal TATA element.

Transcription of the TRP3 gene of Saccharomyces cerevisiae is regulated by GCN4p from a position proximal to the transcriptional initiation sites. The promoter's apparent lack of a conventional TATA element sequence has led it to be used as a model for TATA-less promoters. Through mutational analysis of the TRP3 promoter, we have identified two additional regulatory elements required for expression. The first, located 57 base pairs (bp) upstream of the GCN4p binding site, binds ABF1p in vitro. The ABF1p binding site was required for maximal levels of GCN4p-activated transcription in vivo; however, the -fold activation by GCN4p was not altered by ABF1p. The second element, positioned 23 bp downstream of the GCN4p binding site, contained the TATA-like sequence, TATTAA. This element was required for both basal and activated expression and almost certainly functions as a TATA-binding protein interaction site. Mutations that improved its TATA character for native or an altered specificity mutant of TATA-binding protein correspondingly improved its function. Interestingly, basal expression induced by ABF1p was virtually unchanged in the presence of point mutations in the TATTAA element. Furthermore, unlike the case for HIS3 where only a limited subset of TATA-like sequences can activate transcription in conjunction with GCN4p, many divergent TATA-like sequences allowed GCN4p activation of TRP3. We suggest that the apparent promoter specific use of these TATA elements by GCN4p results from ABF1p amplifying the GCN4p-induced expression to a detectable level.

Characterization of NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription.

The GAL1-10 genes of Saccharomyces cerevisiae are regulated by the interaction of cis- and trans-acting factors which facilitate activated transcription in galactose but not in glucose medium. By selecting mutations that allow expression of a defective gal1-10-his3 hybrid promoter, we have identified a novel gene, NGG1, which is required for glucose repression of the GAL10-related his3-G25 promoter. ngg1 was identified as a recessive null mutation that in the presence of a gal80 background resulted in a 300-fold relief of glucose repression for the his3-G25 promoter. This compared with a 20-fold and negligible relief of repression in gal80 and ngg1 strains, respectively. Deletion analysis of the his3-G25 promoter showed a correlation between the number of GAL4p binding sites and the relative level of NGG1p activity. Relief of glucose repression by NGG1 was dependent on the presence of GAL4, but was independent of the GAL4 promoter. In addition, NGG1p activity was seen for a promoter construct containing independent GAL4p binding sites. These results suggest that NGG1p acts to inhibit GAL4p function in glucose medium. We have cloned NGG1 by complementation and found that it contains an open reading frame of 2106 bp which could encode a protein with a molecular weight of 79,230.

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex.

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.

A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element.

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle.

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.

Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.

Regulation of myocardial Ca2+-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.

Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro.

Regulated transcription by eukaryotic RNA polymerase II (Pol II) requires the functional interaction of multiple protein factors, some of which presumably interact directly with the polymerase. One such factor, the yeast GCN4 activator protein, binds to the upstream promoter elements of many amino acid biosynthetic genes and induces their transcription. Through the use of affinity chromatography involving GCN4- or Pol II-Sepharose columns, we show that GCN4 interacts specifically with Pol II in vitro. Purified Pol II is retained on the GCN4-Sepharose column under conditions in which the vast majority of proteins flow through. Moreover, Pol II can be selectively isolated from more complex mixtures of proteins. Conversely, GCN4 protein, synthesized in vitro or in Escherichia coli, specifically binds to the Pol II-Sepharose column under equivalent conditions. Using deletion mutants, we also show that the DNA-binding domain of GCN4 is both necessary and sufficient for this interaction. We suggest the possibility that this GCN4-Pol II interaction may be important for transcription in vivo.

Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene.

We have isolated two genomic clones which together encode the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. One of the two 16.5 kilobase (kb) genomic inserts in the lambda phage vector Charon 4A contains 23 exons extending from the polyadenylation site at the 3' end of the ATPase gene to within 38 nucleotides of the translation initiation codon in the 5' exon. An overlapping genomic insert of 16.5 kb contains the remainder of the 5' exon and a further 8 kb of upstream sequence. S1 nuclease mapping and primer extension analysis of the 5' end of the Ca2+-ATPase mRNA indicate that the transcription initiation site is located 185 base pairs (bp) upstream of the translation initiation codon. A "TATA" box (CA-TAAA) was found at position -30 and the sequence CCAAT was found at position -78 relative to the transcription initiation site. In a previous study (Brandl, C. J., de Leon, S., Martin, D. R., and MacLennan, D. H. (1987) J. Biol. Chem. 262, 3768-3774) cDNAs for neonatal and adult forms of the fast-twitch Ca2+-ATPase were shown to encode different carboxyl-terminal sequences, presumably as a result of alternative splicing. We have now found that these different DNA sequences encoding different carboxyl-terminal sequences are located in different exons. Exon boundaries of the Ca2+-ATPase gene did not correlate well with proposed domain boundaries for the Ca2+-ATPase protein. The locations of exon/intron boundaries were only partially conserved between the Ca2+-ATPase gene and a Na+/K+-ATPase gene (Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., Allikmets, R. L., Ushkaryov, Y. A., Melkov, A. M., Smirnov, Y. V., Malyshev, I. V., Dulubova, I. E., Petrukhin, K. E., Gryshin, A. V., Sverdlov, V. E., Kiyatkin, N. I., Kostina, M. B., Modyanov, N. N., and Sverdlov, E. D. (1987) FEBS Lett. 213, 73-80) and they did not follow closely the boundaries of amino acid sequences that are highly conserved among a group of ion transport ATPases.