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.

Physiological changes and clinical correlations of dyspnea in cancer outpatients.

The purposes of this cross-sectional study of 75 outpatients of a general oncology clinic were to assess the subjective and objective factors associated with dyspnea in cancer patients and to characterize factors that might contribute to respiratory muscle weakness demonstrated in a previous study. Patients with moderate to severe shortness of breath completed visual analogue scales (VAS) of shortness of breath (SOB) and anxiety; other data were acquired from pulmonary function tests, including maximum inspiratory pressure (MIP) and expiratory pressures; chest radiography; arterial blood gases; measurement of hemoglobin, serum potassium, phosphate, calcium, albumin, and magnesium; and ultrasound study of the diaphragm for thickness and excursion. The correlation coefficient between SOB VAS and anxiety VAS was 0.26 (P = 0.03). In stepwise multiple regression analyses, only the regression coefficient for anxiety remained significant at P < 0.05 in the multivariate model with SOB VAS as the dependent variable. The multivariate model using MIP (a measure of respiratory muscle strength) as the dependent variable, found significance for total diaphragmatic excursion, hemoglobin, phosphate, residual volume over total lung volume, vital capacity, percent predicted total lung capacity, oxygen saturation, and forced vital capacity. The regression coefficients for these variables were significant at P < 0.05 and the model accounted for 58% of the variance of MIP.

Identification of an amino acid substitution in human alpha 1 Na,K-ATPase which confers differentially reduced affinity for two related cardiac glycosides.

The ouabain-resistant cell line H1C1 displays a 30-fold differential of reduced sensitivity to the structurally related cardiac glycosides digoxin and digitoxin (Baker, R. M. (1976) in Biogenesis and Turnover of Membrane Macromolecules (Cook, J.S., ed) pp. 93-103, Raven Press, New York). Since these ligand congeners differ only by the presence of a hydroxyl group at C-12 of digoxin we predicted that the H1C1 phenotype must reflect a mutation which alters the binding site of the cardiac glycoside receptor (Na,K-ATPase). Complementary DNA encoding the alpha 1 Na,K-ATPase was prepared from H1C1 cell total RNA by reverse transcription-coupled polymerase chain reaction and these cDNAs were cloned. Sequence analysis of the reverse transcriptase-polymerase chain reaction clones revealed several independent isolates containing a G > A transition at nucleotide 332 of the propeptide coding sequence, generating the amino acid substitution C108Y. The ability of this substitution to confer differential sensitivity for digoxin and digitoxin was tested and confirmed by expressing a human alpha 1 C108Y-Na,K-ATPase in wild type HeLa cells and assaying for inhibition of cell growth and inhibition of Na,K-ATPase activity. Phenylalanine or alanine substitutions of this cysteine also confer this pattern of ligand discrimination. Ouabain-resistant Na,K-ATPase substitutions, at positions other than Cys-108 failed to exhibit differential sensitivity indicating that this ligand discrimination is unique to Cys-108 substitutions rather than a general property of cardiac glycoside-resistant mutants. It is proposed that differential resistance of the C108Y receptor for these ligands is a consequence of altering two features of the ligand-receptor interaction; one, a disruption of a common hydrogen bond resulting in general loss of affinity for cardiac glycosides and the other, formation of a new H-bond between the C-12 hydroxyl of digoxin and the receptor, specifically augmenting the stability of this ligand-receptor complex.

Different levels of regulation accomplish the switch from type II to type I collagen gene expression in 5-bromo-2'-deoxyuridine-treated chondrocytes.

The shift of chick embryo chondrocytes to a fibroblastic phenotype by 5-bromo-2'-deoxyuridine (BrdUrd) has been used to examine the molecular basis of the switch from type II to type I collagen gene expression. Transcription rates of each of these three collagen genes before and after this shift were measured in nuclear run-on transcription assays with double-stranded 3'-cDNA probes specific for each of these three mRNAs. Degradation rates of each of these RNAs were calculated from the rate of decrease in the concentration of each RNA after the inhibition of synthesis with actinomycin D. The shut-off of the expression of the type II collagen gene during this shift was shown to occur at the transcriptional level, since the transcription rate of this gene decreased dramatically. The decay rate of the type II mRNA (half-life of approximately 15 h) is not significantly faster in BrdUrd-treated cells. The alpha 1(I) gene is transcribed at similar rates in untreated and shifted chondrocytes, but the steady state level of alpha 1(I) RNA in chondrocytes is only 1.5% of that in shifted cells. Although the measured degradation rate of the total alpha 1(I) RNA from untreated chondrocyte cultures is approximately the same as in shifted cells (half-life of approximately 12 h), indirect evidence suggests that this alpha 1(I) RNA is derived from a low level of fibroblast contamination of these chondrocyte cultures. The alpha 1(I) RNA synthesized by untreated chondrocytes is assumed therefore to be broken down very rapidly in the nucleus. The alpha 2(I) gene is also transcribed in untreated chondrocytes at rates similar to shifted cells but, unlike alpha 1(I) RNA, its steady state level in untreated chondrocytes is approximately 30% of its level in shifted chondrocytes. The increased level of alpha 2(I) RNA in shifted cells may be regulated in part by an increase in stability of the alpha 2(I) mRNA, which has half-lives of 5.2 and 10.4 h, respectively, in untreated and shifted chondrocytes. The alpha 2(I) RNA in the untreated chondrocytes was found to have a different 5' end from that present in the BrdUrd-shifted chondrocytes or in chick embryo fibroblasts. The presence of this altered RNA in untreated chondrocytes explains the absence of synthesis of the fibroblastic alpha 2(I) collagen polypeptide chains in these chondrocytes, despite the presence of the alpha 2(I) RNA as measured with 3' probes.

Estimation of relationship coefficients among progeny derived from wind-pollinated orchard seeds.

Statistical analyses of trees produced from wind-pollinated seeds to determine levels of additive variance need to be adjusted to compensate for the relationships among the progeny. The assumption that the coefficient of relationship among the progeny is 0.25 will lead to inaccurate estimation of the additive variance if the relationships among the progeny are not entirely half-sib. Foreign pollen intrusion into the orchard pollen cloud, selfing, and reproductive phenology variation (i.e., mating proportion differences) among the parent trees may lead to varying proportions of self, self-half, half-sib, and full-sib relationships among the progeny. The variance of the coefficient of relationship from 0.25 for two coniferous seed orchards subjected to simulated selfing and foreign pollen problems illustrate the need for caaution when estimation techniques are employed.

Conservation of an AE3 Cl-/HCO3- exchanger cardiac-specific exon and promoter region and AE3 mRNA expression patterns in murine and human hearts.

A cardiac-specific variant of the rat AE3 Cl-/HCO3- exchanger mRNA is transcribed from a promoter located in the sixth intron of a larger transcription unit expressed in brain and other tissues. The cardiac mRNA contains an alternative first exon encoding a 73-amino acid NH2-terminal sequence that replaces the first 270 amino acids of the brain AE3 variant. The present study was conducted to determine whether the cardiac-specific promoter region and exon are conserved in other species and to examine the expression patterns of AE3 mRNAs in adult tissues and during development. Analysis of murine and human genomic clones showed that both contain counterparts of the rat alternative exon. The cardiac promoter region is highly conserved in all three species and contains several potential transcription factor binding sites, including consensus MCAT and E-box sequences. Tissue-specific and developmental patterns of AE3 gene expression were examined by Northern blot hybridization. Mouse and human, like the rat, express both the 3.6-kb cardiac-specific AE3 mRNA and a 4.4-kb AE3 transcript found in brain, heart, and other tissues. Levels of the cardiac-specific transcript in mouse heart increase 17-fold between the fetal and adult stages, while the amount of the longer AE3 mRNA in heart decreases substantially. Furthermore, although the cardiac-specific mRNA is expressed in both atria and ventricles of mouse heart, the longer transcript is confined to the atria. These results suggest that the two AE3 variants have distinct roles in cardiac function and that the mechanisms regulating their expression are different.

Modulation of pump function by mutations in the first transmembrane region of Na(+)-K(+)-ATPase alpha 1-subunit.

The Cys in the first transmembrane region of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) alpha 1-subunit has been shown to be a critical determinant of cardiac glycoside binding. To study the role of this Cys on ion transport activity, we measured pump currents in HeLa cells expressing wild-type or mutant alpha 1-subunit cDNAs. The endogenous ouabainsensitive Na(+)-K(+)-ATPase was selectively inhibited by growing the cells in 0.1 microM ouabain. A Cys-to-Tyr substituted mutant exhibited decreased sensitivity to digitoxin but not digoxin compared with wild type. The decreased affinity for digitoxin was due to a faster dissociation rate. In contrast, the Cys-to-Ala substitution did not significantly alter the sensitivity to digitoxin or digoxin. Both wild-type and mutant cells displayed marked external K(+)-dependent pump currents; however, the affinity for K+ was reduced by the mutations. The decrease in K+ affinity was due to a slower association rate. The results show that the Cys that interacts with cardiac glycosides also participates in the sensitivity of the pump to external K+.

In situ localization of sodium-potassium ATPase mRNA in developing mouse lung epithelium.

The ontogeny of Na(+)-K(+)-adenosinetriphosphatase (ATPase) mRNA in the mouse lung was examined, using alpha- and beta-isoform-specific probes in Northern blot assays and for in situ hybridization analysis. Northern blot assays demonstrated an increase in Na(+)-K(+)-ATPase expression in the perinatal period, peaking at birth (D1), with alpha 1- and beta 1-isoform levels reaching six to eight times adult levels. In situ alpha 1-isoform hybridization signals were localized primarily to developing airway epithelium and were most intense on D1. Postnatally, alpha 1-isoform hybridization signals persisted in airway epithelium, although progressively diminishing in intensity relative to perinatal levels. In developing alveolar regions, alpha 1-isoform hybridization signals remained slightly above background during this period. beta 1-Isoform hybridization signals increased dramatically during the perinatal period in both developing airway and alveolar epithelia. Postnatally, beta 1-isoform hybridization signals declined slightly in airway epithelium and developed a punctate pattern in alveolar epithelium. These data indicate that the perinatal increase in Na(+)-K(+)-ATPase expression observed in the developing mouse lung is localized primarily to epithelial structures and is therefore likely to be related to the changes in transepithelial ion and fluid transport known to occur in the perinatal period.

Association of a change in chromatin structure with a tissue-specific switch in transcription start sites in the alpha 2(I) collagen gene.

Chick embryonic sternal chondrocytes do not synthesize alpha 2(I) collagen until they are shifted by treatment with 5-bromo-2'-deoxyuridine (BrdUrd) to a fibroblastic phenotype, yet they transcribe this gene as rapidly as BrdUrd-treated cells. To examine further this transcription, the DNase I hypersensitive sites were mapped in the 5' region of this gene in chondrocytes, BrdUrd-treated chondrocytes, fibroblasts and three types of non-transcribing cells. A DNase I hypersensitive site at -200 bp, previously shown to be associated with the active transcription of this gene in fibroblasts, is not present in chondrocyte chromatin. The chondrocyte alpha 2(I) gene contains, however, a novel major hypersensitive site in the DNA region corresponding to the fibroblast intron 2, near the chondrocyte-specific transcription initiation site of this gene. This novel hypersensitive site is associated with the use of this alternate start site by chondrocytes, since it is lost when BrdUrd treatment causes these chondrocytes to switch to the initiation of transcription at the fibroblast start site. The BrdUrd-treated chondrocytes contain the same alpha 2(I) hypersensitive sites as fibroblasts, except that fibroblasts have an additional, previously unreported, site at -1000 bp.

Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Inhibition of cardiac delayed rectifier K+ current by overexpression of the long-QT syndrome HERG G628S mutation in transgenic mice.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.