Marcia M. Shull

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.

Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders.

To better understand the formation of the cardiovascular system and its disease states, models amenable to manipulation must be developed. In this article we present two models. One is a small animal model for an inflammatory disorder that can lead to heart failure. Production of this model is based on the ability of blastocyst-derived embryonic stem cells, which can be genetically altered in vitro by a technique called gene targeting, to reconstitute an entire animal when reintroduced into a blastocyst and allowed to colonize the germ line of the resulting chimeric embryo. The other model is based on the capacity of embryonic stem cells to differentiate in culture into embryo-like structures called embryoid bodies. Embryoid bodies contain angioblasts, or prevascular endothelial cells, which can be induced to undergo aspects of vascular development by manipulation of culture conditions.

Characterization of two genes for the human Na,K-ATPase β subunit

Abstract A total of 29 human genomic DNA clones that hybridize with cDNAs for the sheep and rat Na,K-ATPase β subunits have been isolated, classified by restriction endonuclease mapping and Southern blot hybridization analysis, and sequenced. One class of clones, designated ATP1BL1, represents a processed pseudogene for the β subunit. The second class, designated ATP1B, includes 15 overlapping genomic clones and represents a functional gene for the human Na,K-ATPase β subunit. ATP1B spans about 26.7 kb of genomic DNA and includes 24 kb of intron sequence. The complete mRNA transcript for the human β subunit is encoded by six exons, ranging in size from 81 to 1427 bp. Primer extension and S1 nuclease protection experiments with human kidney RNA indicate the presence of two major transcription initiation sites at −510 and −201 to −191, with minor initiation sites at −268, −182 to −174, and −142. The distal initiation site at −510 is preceded by consensus sequences for CAAT and TATA boxes. The DNA sequence preceding the proximal heterogeneous initiation sites contains a CAAT box, but no TATA box. Two of the 12 GC boxes (GGCGGG and CCCGCC) located in the 5′ region of ATP1B are located between this CAAT box and the proximal clusters of transcription initiation sites.

Discordant segregation of Na+, K+-adenosine triphosphatase alleles and essential hypertension

OBJECTIVES To determine whether the alpha 2 and or beta 1 isoforms of the Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) are involved in the pathogenesis of essential hypertension. DESIGN Segregation analysis of polymorphic DNA markers was used to test the involvement of Na+,K(+)-ATPase in essential hypertension. PARTICIPANTS Children with persistent hypertension having one parent with essential hypertension were included in the study. Criteria for persistent hypertension were blood pressure readings with systolic and/or diastolic levels exceeding the 95th percentile based upon age and sex. The diagnosis of hypertension for adults, including parents and older siblings, was confirmed using criteria recommended in the 1988 report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. RESULTS In three essential hypertensive families consisting of 18 members including 11 hypertensives, several obligate recombinants between the Na+,K(+)-ATPase alpha 2 isoform marker and the hypertension phenotype were observed. Similarly, in one hypertension family consisting of four members, obligate recombinants between the beta 1 isoform marker and the disease were observed. CONCLUSIONS The discordant segregation of the alpha 2 and beta 1 isoform markers and essential hypertension suggests that neither the Na+,K(+)-ATPase alpha 2 nor beta 1 isoform genes play a primary role in the pathogenesis of hypertension in the families studied.

Phenotypes of TGFß Knockout Mice

The transforming growth factor-s (TGFs) family consists of structurally related polypeptides that mediate important physiological processes. Significant advances have been made recently in understanding the functions of these molecules in vivo by the targeted ablation of various members of the super gene family. The family members include the TGF(is, activins, inhibins, bone morphogenetic proteins, Mullerian-inhibiting substance, nodal, dorsalin,and the products of the Drosophila decapentaplegic, and Xenopus Vg-1 genes (for review see refs. 1–4). Recently, the growth differentiation factor (GDF) family of proteins, a new member of the TGFβ family, has been described (5–7). TGFβl was first isolated from human platelets (8) and the cDNA sequence was later determined (9). TGFs 1 is a disulfide-linked homo-dimer of two 112 amino acid chains. Each chain is synthesized as the C-terminal domain of a 390 amino acid precursor. It has the characteristics of a secretory polypeptide, contains a hydrophobic signal sequence for translocation across the endoplasmic reticulum, and is glycosylated. The precursor cleavage site is a sequence of four basic amino acids immediately preceding the bioactive domain (9).