The cardiac glycoside binding site on the Na,K-ATPase alpha2 isoform plays a role in the dynamic regulation of active transport in skeletal muscle.

The physiological significance of the cardiac glycoside-binding site on the Na,K-ATPase remains incompletely understood. This study used a gene-targeted mouse (alpha2(R/R)) which expresses a ouabain-insensitive alpha2 isoform of the Na,K-ATPase to investigate whether the cardiac glycoside-binding site plays any physiological role in active Na(+)/K(+) transport in skeletal muscles or in exercise performance. Skeletal muscles express the Na,K-ATPase alpha2 isoform at high abundance and regulate its transport over a wide dynamic range under control of muscle activity. Na,K-ATPase active transport in the isolated extensor digitorum longus (EDL) muscle of alpha2(R/R) mice was lower at rest and significantly enhanced after muscle contraction, compared with WT. During the first 60 s after a 30-s contraction, the EDL of alpha2(R/R) mice transported 70.0 nmol/g.min more (86)Rb than WT. Acute sequestration of endogenous ligand(s) in WT mice infused with Digibind to sequester endogenous cardiac glycoside(s) produced similar effects on both resting and contraction-induced (86)Rb transport. Additionally, the alpha2(R/R) mice exhibit an enhanced ability to perform physical exercise, showing a 2.1- to 2.8-fold lower failure rate than WT within minutes of the onset of moderate-intensity treadmill running. Their enhanced exercise performance is consistent with their enhanced contraction-induced Na,K-ATPase transport in the skeletal muscles. These results demonstrate that the Na,K-ATPase alpha2 isozyme in skeletal muscle is regulated dynamically by a mechanism that utilizes the cardiac glycoside-binding site and an endogenous ligand(s) and that its cardiac glycoside-binding site can play a physiological role in the dynamic adaptations to exercise.

Chromatin fine-structure mapping of the goat beta F gene in fetal erythroid tissue.

Using a restriction enzyme accessibility assay, we have previously demonstrated that the chromatin structure immediately proximal to the goat beta F-, beta C-, and beta A-globin genes changes in a manner which parallels their developmentally regulated expression. More specifically, the PvuII recognition sequence, located 9 nucleotides upstream from the transcriptional start site in each of the three genes, is accessible to digestion only in nuclei prepared from erythroid tissue in which the respective gene product is expressed. Here we describe two restriction enzyme sites further upstream from the transcription start sites (HindIII at -700 and SacI at -480) which were not accessible to digestion in fetal erythroid nuclei. Conversely, two sites within the second coding block of the beta F gene (AccI at +276 and BamHI at +470) were accessible in fetal erythroid tissue. The corresponding sites in the beta C and beta A genes were not available for digestion in the same fetal tissue. Processive exonuclease III digestion in situ from the three accessible restriction enzyme sites in the beta F gene allowed us to define more closely the limits of these open regions. Resistance to exonuclease III digestion was encountered at or near both intron-exon junctions flanking the first intervening sequence of the beta F gene. Conversely, no resistance to exonuclease III digestion was evident in either the first or second coding blocks or the 5' untranslated region. Digestion upstream from the PvuII site of the beta F gene was negligible. High-resolution mapping by S1 nuclease analysis indicated that the endpoint of exonuclease III digestion from this site lay immediately downstream of the ATA box.

Identification of a recently evolved goat embryonic beta-globin pseudogene which retains transcriptional activity in vitro.

A clone containing the entire goat epsilon V beta-globin gene, which lies downstream from the two tandemly duplicated four-gene sets containing the beta C and beta A genes in the linkage group 5'-epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-3', was isolated, and the sequence of the gene was determined. epsilon V is most homologous to the first gene in each of these sets, epsilon I and epsilon III, and appears to be a third duplicated copy of these genes, possibly the first gene in a third four-gene set. Homology of epsilon V to epsilon I is very high (93.2%) in coding regions, and all transcription, processing, and potential translation consensus sequence elements appear to be present, although the Hogness box of epsilon V is altered compared with that of epsilon I by the deletion of an A(AATAAAA----AATAAA). Nevertheless, epsilon V is clearly a pseudogene as a result of two deletions and one insertion (or insertion-deletion) in its coding sequence, the first of which produces an in-frame stop codon at amino acid 54. Unlike the more highly mutated goat beta-like pseudogene duplicates psi beta X and psi beta Z, epsilon V acquired its defects after the duplication event in which it was created. Its recently acquired defects have left the epsilon V promoter sufficiently conserved to retain transcriptional activity in vitro. The acquisition of defects by this gene may be related to the multiple gene duplications which have created at least five epsilon type genes in the goat beta-globin locus.

Polymorphism in an androgen-regulated mouse gene is the result of the insertion of a B1 repetitive element into the transcription unit.

The single-copy RP2 gene in mice produces three major mRNAs, the abundances of which are significantly increased in the kidneys by the administration of testosterone. S1 nuclease analysis of the kidney mRNAs indicated that they differ in the lengths of their 3' untranslated regions as a result of the use of different polyadenylation sites. When the mRNAs from different inbred mouse strains were examined by Northern blot analysis, it was observed that the largest mRNA varies in size, whereas the sizes of the other mRNAs remain the same. In DBA/LiHa and DBA/2J mice, the largest mRNA is approximately 2,150 nucleotides long, whereas the corresponding mRNA in C57BL/6J and BALB/cJ mice is only 1,950 nucleotides in length. All of these strains also have RP2 mRNAs that are 1,450 and 1,350 nucleotides long. By S1 nuclease mapping and comparison of the sequence of cDNA clones representing these mRNAs in DBA/LiHa and C57BL/6J mice, we determined that this size difference or polymorphism observed in the largest mRNA is the result of the insertion of a member of the B1 family of repeats into the 3' untranslated region of the RP2 gene in DBA mice. This particular B1 repeat is transcribed by RNA polymerase III in vitro, and its transcriptional orientation is opposite to that of the RP2 transcript. The polymorphism described here is evidence for the mobility of B1 repetitive elements within the genome.

Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders.

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.

We have identified and characterized the gene for a novel zinc finger transcription factor which we have termed lung Krüppel-like factor (LKLF). LKLF was isolated through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe and is closely related to this erythroid cell-specific gene. LKLF is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen. The gene is developmentally controlled, with expression noted in the 7-day embryo followed by a down-regulation at 11 days and subsequent reactivation. A high degree of similarity is noted in the zinc finger regions of LKLF and EKLF. Beyond this domain, the sequences diverge significantly, although the putative transactivation domains for both LKLF and EKLF are proline-rich regions. In the DNA-binding domain, the three zinc finger motifs are so closely conserved that the predicted DNA contact sites are identical, suggesting that both proteins may bind to the same core sequence. This was further suggested by transactivation assays in which mouse fibroblasts were transiently transfected with a human beta-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human beta-globin promoter containing the CACCC sequence previously shown to be a binding site for EKLF. Mutation of this potential binding site results in a significant reduction in the reporter gene expression. LKLF and EKLF can thus be grouped as members of a unique family of transcription factors which have discrete patterns of expression in different tissues and which appear to recognize the same DNA-binding site.

Expression of human beta-globin genes in transgenic mice: effects of a flanking metallothionein-human growth hormone fusion gene.

In an attempt to place a human beta-globin gene in an open chromatin domain regardless of its site of integration in the mouse genome, we microinjected into fertilized mouse eggs a construct in which the human beta-globin gene and a mouse metallothionein-human growth hormone fusion gene were juxtaposed and oriented in opposite directions. Mice that developed from injected eggs and that grew larger than normal were analyzed for human beta-globin mRNA. The globin genes were not expressed in erythroid tissue but were expressed with the same tissue specificity as metallothionein-human growth hormone. These results suggest that sequences which control metallothionein-human growth hormone gene expression are capable of stimulating the expression of a flanking gene in an orientation-independent and tissue-specific manner. As a control for this experiment, we deleted the metallothionein-human growth hormone transcription unit and noted that the human beta-globin gene then was expressed at high levels with erythroid tissue specificity.

Regulated expression of the human beta globin gene in transgenic mice requires an upstream globin or nonglobin promoter.

Transgenic mice have been used extensively to study elements governing the erythroid-specific developmental switch from human fetal gamma to human adult beta globin. Previous work demonstrated that a small construct composed of hypersensitive site 2 (HS2) of the locus control region (LCR) linked to the gamma and beta globin genes (HS2-gamma-beta) is sufficient for correct tissue and temporal expression of these genes, whereas HS2-beta alone is inappropriately expressed in the embryo. Two models, which are not mutually exclusive, have been proposed to explain these results and those of other constructs in transgenic mice. One model emphasizes the conserved polarity in the globin locus and suggests a distance effect whereby the beta globin gene must be removed from the LCR/HS2 to prevent an early and incorrect activation of this gene in the embryonic compartment. A second hypothesis proposes a competition between the gamma and beta globin gene promoters for interaction with the LCR/HS2. The active gamma globin gene promoter positioned between the LCR/HS2 and the beta globin gene thereby interacts with the HS2 elements early in erythroid development and is expressed until a change in putative stage-specific nuclear factors makes an interaction with the adult beta globin gene more favorable. In an effort to test the competition model, a construct has been prepared in which a small deletion was produced in the promoter region of the gamma globin gene while in the context of the HS2-gamma-beta plasmid. Analysis of this construct in transgenic mice reveals a constitutive unregulated expression of the human beta globin gene during erythroid development. To determine if this competition effect is specific for globin genes, a heterologous reporter gene has been substituted for the gamma globin gene in the construct HS2-gamma-beta. In this case, the beta globin gene exhibits correct developmental expression. This data is consistent with a model in which transcription from a promoter upstream of the beta globin gene in some manner protects this adult gene from activation by the LCR/HS2 during early development.

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Molecular weight determinations on the alpha-globin and beta-globin mRNAs.

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster.

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.

Sequence and linkage of the goat epsilon I and epsilon II beta-globin genes.

Overlapping clones containing beta-globin genes have been isolated from a goat genomic library which establish the linkage arrangement 5'-epsilon I-epsilon II-psi beta X-beta C-3'. The complete nucleotide sequence of the epsilon I and epsilon II genes was determined. The sequences of these two genes, along with those previously reported for psi beta X and beta C, complete the sequence of the genes of this linkage set. The first gene in the quadruplet, epsilon I, shows unexpectedly high homology with the human epsilon globin gene both in coding and non-coding regions, and encodes a globin protein that is 90% homologous to human epsilon. The only major difference between the goat epsilon I gene and the human epsilon gene is the presence of an insertion element in the second intron of epsilon I. This element is repetitive in nature and is similar to those found in the second intron of the gamma, beta C and beta A globin genes of the goat. epsilon II also shows high nucleotide homology to the human epsilon globin gene in coding regions and encodes a protein 79% homologous to human epsilon. Notably, however, epsilon II has equivalent nucleotide homology in coding regions to the gamma and epsilon genes of the human locus. The insertion element present in epsilon I is not present in epsilon II. A comparison of the goat beta globin set described here, based on linkage arrangement, nucleotide homology and divergence analysis indicates that this subset of goat beta globin genes is analogous to the entire beta globin loci of other mammalian species. These analyses further indicate that the embryonic genes in these clusters are evolving more slowly than the adult beta globin genes. Comparison of the 5' flanking sequences of epsilon I and epsilon II with those of the beta-embryonic globin genes of other mammals reveals a conserved sequence, C-A-C-C-C-C-T-G, located 28 to 29 bases upstream from the C-C-A-A-T consensus sequence, which appears at this position in the embryonic genes, but in none of the non-embryonic genes. Significantly, this sequence is selectively conserved in the human alpha embryonic globin gene, zeta, which diverged from the beta embryonic genes 500 million years ago, and it may therefore represent an embryonic recognition or signal sequence.

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo.

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.

Differential regulation of rat T-kininogen by tumor necrosis factor and interleukin-6.

Rat T-kininogen (T-KG), a cysteine protease inhibitor, is an acute phase reactant which is induced to high levels in response to inflammation. Both hormones and cytokines participate in this regulation. To investigate the cis-acting elements responsible for the induction of gene expression, various 5'-fragments of the rat T-KG gene were fused to a chloramphenicol acetyltransferase marker gene. These constructs were transfected into a rat hepatoma cell line which was then treated with tumor necrosis factor or interleukin-6 or both cytokines. Expression of the chloramphenicol acetyltransferase gene was induced with interleukin-6 treatment, but suppressed by tumor necrosis factor. The 5'-region of the T-KG gene responsible for conferring both of these effects was localized between nucleotides -404 to -210 upstream of the transcription start site. Fragments containing this region were found to be effective in either orientation, and could also regulate a heterologous promoter.

Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts.

OBJECTIVE: The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity. METHODS: For contractility studies, a total of 57 trabeculae were isolated from two non-failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three alpha isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. RESULTS: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p << 0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol.g-1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three alpha isoforms were expressed at the mRNA level in both normal and failing hearts. CONCLUSIONS: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.

Structural and functional analysis of the goat epsilon-globin genes.

Since none of the vertebrate beta-globin loci studied to date has more than two functional embryonic beta-like globin genes, it would be unique if all six goat embryonic beta-globin genes were required for its survival. In this study we have asked whether all six embryonic genes in the goat are functional. This question has been addressed by examining the transient expression of these genes in HeLa cells and correlating these results with the sequence information obtained to date. Our studies show that only epsilon I and epsilon II are functional while the remaining four epsilon-globin genes are nonfunctional, i.e., pseudogenes. Interestingly, the two active epsilon-globin genes are located at the 5' end of the locus. While this unusual inactivation pattern may be the result of chance, it could also have resulted because the two duplication events, of the ancestral gene set epsilon-epsilon-psi beta-beta, did not include distally located regulatory region(s) essential for epsilon-globin gene expression. Once separated from the 5'-regulatory sequences the remaining four embryonic genes (epsilon III, epsilon IV, epsilon V and epsilon VI) accumulated mutations and became pseudogenes.

cDNA cloning, mapping and expression of the mouse propionyl CoA carboxylase beta (pccb), the gene for human type II propionic acidaemia.

Propionyl CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of amino acids, odd-chained fatty acids and other metabolites. PCC is composed of two equal subunits, alpha and beta, which are encoded by two separate genes at two distinct human loci. Mutations of either gene in humans results in propionic acidemia (PA). To identify the mouse cDNA for the propionyl CoA carboxylase beta-subunit (pccb), we have screened the mouse EST database using the human sequence. The murine mRNA transcript is approximately 2.3 kb, nearly 500 bps larger than the human approximately 1.8 kb transcript. A PAC genomic DNA clone from the mouse was also isolated and used to generate probes and PCR primers for mapping the pccb locus in the mouse. Both the C57Bl/6JEi and Spret/Ei alleles for regions flanking the pccb gene were sequenced to identify RFLPs. The Jackson Laboratory BSS and BSB backcross panel DNAs were then analyzed using a DdeI polymorphism placing the pccb locus on mouse chromosome 9. Northern blots of adult tissue show that the pccb gene is broadly expressed in the mouse. The approximately 2.3 kb transcript is most abundantly expressed in the kidney, liver, small intestine and stomach tissues.

Primary structure of a gene encoding rat T-kininogen.

A gene encoding the acute-phase reactant, T-kininogen, has been isolated from a rat genomic library. The nucleotide sequence of all exons as well as of a large portion of the introns was determined. The gene, T-KG, is approximately 24 kb in length and contains 11 exons. The promoter appears to be rather unusual in that there are no consensus CCAAT or TATAA boxes or SP1-binding sites. An A-rich region present upstream of the transcription start point may function as a TATAA-equivalent sequence. In addition, several potential glucocorticoid regulatory elements and a sequence similar to the AP-1 binding site are present in the 5' region of the gene. The intron/exon boundaries in this T-KG gene are identical to those seen in the human K-kininogen gene. This is consistent with the inclusion of T-KG in the cystatin supergene family of cysteine protease inhibitors.