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J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 109 records · Page 6Linked to original sources
PubMed1989

A comparison of the beta A-and beta B-globin gene clusters of sheep.

Domestic sheep have two common alleles at the adult beta-globin locus, beta A and beta B. Here we report the structure of the beta-globin locus of A-haplotype sheep. The locus consists of 12 genes, organized as a triplicated 4-gene set: 5' epsilon 1-epsilon II-psi beta I-beta C-epsilon III-epsilon IV-psi beta II-beta A-epsilon V-epsilon VI-psi beta III-beta F 3'. This arrangement is identical to that of the closely related goat locus. Sheep with the B haplotype have a locus arrangement consisting of a duplicated four-gene set, lacking the beta C gene as well as three other genes present in A sheep and goats. In order to understand the evolutionary history of the B sheep locus, we have sequenced the beta B gene from these sheep, and the beta C gene from A-haplotype sheep, and compared the sequences to those of the sheep beta A, goat beta C, and beta A, and cow adult beta genes. Our results indicate that the beta B gene has diverged recently from the beta A gene, and therefore the beta B locus structure may have resulted from a recent deletion from a triplicated locus.

Amino Acid Sequence↗
PubMed1989

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

A total of 29 human genomic DNA clones that hybridize with cDNAs for the sheep and rat Na,K-ATPase beta 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 beta subunit. The second class, designated ATP1B, includes 15 overlapping genomic clones and represents a functional gene for the human Na,K-ATPase beta subunit. ATP1B spans about 26.7 kb of genomic DNA and includes 24 kb of intron sequence. The complete mRNA transcript for the human beta 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.

Amino Acid Sequence↗
PubMed1989

Analysis of possible repressor elements in the 5'-flanking region of the human beta-globin gene.

Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.

Animals↗
PubMed1989

Sites I and II upstream of the A gamma globin gene bind nuclear factors and affect gene expression.

Five broad regions in and near the A gamma globin gene specifically bind proteins in K562 nuclear extracts. Two are located 5' of the gene, one between -1349 and -1094, and the other between -384 and +52. Each of the two introns has a binding region, +184 to +284 and +930 to +1209. The fifth binding region is within the A gamma globin gene enhancer, a 750 bp Hind III fragment located 3' to the gene. The nuclear factors which bind to any or all of these regions may be important for control of regulation of the A gamma globin gene. Since HPFH point mutations occur at -175, -196 and -202, we investigated K562 nuclear factor binding to this region more closely. Footprints were obtained for two binding sites, one from -168 to -185 (site II) and the other from -267 to -293 (site I). Site II contains an octamer sequence (ATGCAAAT) important for protein binding and expression of the histone H2b and immunoglobulin genes. The HPFH mutation at -175 changes the T in the octamer sequence to a C. Site I is upstream of the bases affected by HPFH mutations. A A gamma globin gene promoter - CAT reporter fusion gene was constructed with a clustered - base substitution of site I or a T----C point mutation at -175. The mutation in site I decreases CAT expression 20X compared to wild-type in erythroid and non-erythroid cells. Site I probably binds a positive regulator. The T----C change at -175 increases expression 2-3X over wild-type in erythroid cells, but not in non-erythroid cells. This increase correlates with the effect of the naturally occurring HPFH, and may result from decreased binding of a negative effector, and/or increased positive factor binding.

Base Sequence↗
PubMed1988

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation.

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.

Animals↗
PubMed1988

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs.

The developmental expression of the multiple isozymes of Na,K-ATPase in rat brain, heart, lung, kidney, and skeletal muscle from fetal (14 days gestation) to adult (55 days) was investigated at the molecular level with cDNA probes specific for the multiple catalytic alpha isoform (alpha 1, alpha 2, alpha 3) and beta subunit mRNAs. Northern and RNA slot blot analyses revealed that these mRNAs are regulated in a tissue-specific manner. The multiple alpha isoform and beta subunit mRNAs appear to be regulated coordinately during ontogenesis with maximum expression occurring between 15 and 25 days of age for brain, heart, kidney, and skeletal muscle, whereas peak expression in lung was observed between 2 and 4 days of neonatal life. Brain tissue showed between 10- and 17-fold increases in the levels of expression for the three individual alpha isoform mRNAs. The alpha 3 mRNA was found to be the predominant alpha isoform transcript in fetal as well as adult brain. Examination of heart tissue showed alpha 1 mRNA to be the major catalytic subunit during development. However, a developmentally regulated transition in alpha 2 and alpha 3 mRNA expression was observed in heart between 7 and 14 days after birth. The alpha 3 mRNA was expressed primarily in fetal and neonatal heart tissue, while alpha 2 mRNA was expressed in juvenile and adult tissue. In kidney and lung, alpha 1 mRNA was the predominant alpha isoform transcript showing temporary increases in expression of 2- and 4-fold, respectively, during development. In contrast to the other tissues, muscle expressed predominantly alpha 2 mRNA following birth, the levels increasing approximately 89-fold during myogenesis. Thus, each tissue examined exhibits a distinct pattern of expression for the Na,K-ATPase catalytic alpha isoforms during ontogenesis.

Aging↗
PubMed1988

Chromosomal localization of human Na+, K+-ATPase alpha- and beta-subunit genes.

Na+, K+-ATPase is a heterodimeric enzyme responsible for the active maintenance of sodium and potassium gradients across the plasma membrane. Recently, cDNAs for several tissue-specific isoforms of the larger catalytic alpha-subunit and the smaller beta-subunit have been cloned. We have hybridized rat brain and human kidney cDNA probes, as well as human genomic isoform-specific DNA fragments, to Southern filters containing panels of rodent X human somatic cell hybrid lines. The results obtained have allowed us to assign the loci for the ubiquitously expressed alpha-chain (ATP1A1) to human chromosome 1, region 1p21----cen, and for the alpha 2 isoform that predominates in neural and muscle tissues (ATP1A2) to chromosome 1, region cen----q32. A common PstI RFLP was detected with the ATP1A2 probe. The alpha 3 gene, which is expressed primarily in neural tissues (ATP1A3), was assigned to human chromosome 19. A fourth alpha gene of unknown function (alpha D) that was isolated by molecular cloning (ATP1AL1) was mapped to chromosome 13. Although evidence to date had suggested a single gene for the beta-subunit, we found hybridizing restriction fragments derived from two different human chromosomes. On the basis of knowledge of conserved linkage groups on human and murine chromosomes, we propose that the coding gene ATP 1B is located on the long arm of human chromosome 1 and that the sequence on human chromosome 4 (ATP 1BL1) is either a related gene or a pseudogene.

Animals↗
PubMed1988

Structural organization of the beta-globin locus of B-haplotype sheep.

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta-globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV-psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta-globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.

Amino Acid Sequence↗
PubMed1988

Multiple forms of the Na,K-ATPase: their genes and tissue specific expression.

The use of genetic tools has been invaluable for examining the potential number of alpha and beta subunits of the Na,K-ATPase. To date at least five genes corresponding to the alpha subunit have been described. Two of these encode the alpha and alpha+ isoforms while the third encodes the novel alpha III subunit. The products of the other two genes have not yet been described; these genes may be pseudogenes or may encode new alpha isoforms or related transport ATPases. The determination of the tissue distribution of the various isoforms of the alpha subunit using Northern blot and slot blot analyses has yielded several interesting findings. Each tissue differs in the abundance and combination of isoforms. While alpha is the major isoform mRNA in kidney, alpha+ mRNA predominates in skeletal muscle. All three alpha subunit mRNAs are present in brain in roughly equal amounts. The newly discovered alpha III isoform mRNA is found not only in brain but in low amounts in stomach and lung. It is possible that individual cell types may contain a defined combination of isoforms which allow the Na,K-ATPase to fulfill its specific physiological role in those cells. It will be interesting to compare the functional properties, i.e., affinity for Na+ and K+, turnover number, response to external changes in environment and response to effector molecules for the enzymes containing each of the alpha subunit isoforms.

Animals↗
PubMed1987

Tissue distribution of mRNAs encoding the alpha isoforms and beta subunit of rat Na+,K+-ATPase.

The tissue distribution of the multiple forms of rat Na+,K+-ATPase was examined at the molecular level with cDNA probes specific for the alpha, alpha (+), alpha III and beta subunit mRNAs. Northern and slot blot analyses demonstrate that these mRNAs are produced in a tissue-specific manner. RNAs encoding the alpha (+) isoform are detected in kidney, brain, heart, adipose, muscle, stomach and lung, whereas alpha III RNA is detected in brain, stomach and lung. Both alpha and beta mRNAs are present in all the tissues studied, although at very different levels. Examination of heart tissue in greater detail demonstrates that the levels of mRNA encoding the alpha subunit are greater in the atria than in the ventricles, while the converse is true for alpha (+).

Animals↗
PubMed1987

Multiple mRNAs from rat kidney and brain encode a single Na+,K+-ATPase beta subunit protein.

To determine whether multiple forms of the Na+,K+-ATPase beta subunit exist in rat kidney and brain, we constructed cDNA libraries from both tissues and isolated clones carrying inserts coding for this subunit. Characterization of these cDNAs by restriction endonuclease mapping and DNA sequencing suggests that a single form of the beta subunit protein is present in these tissues. We did, however, find that the beta subunit was encoded by multiple mRNAs which can be grouped into classes based on the lengths of their 5' and 3' untranslated regions. Size variation at the 3' end is due to the use of at least two of the five potential polyadenylation sites, whereas variation at the 5' end appears to be due to the use of different initiation sites. Northern blot analysis demonstrates the presence of multiple mRNA species which correlate with the sizes observed for the different cDNA classes and also demonstrates that the larger mRNA species are present at different levels in kidney and brain. S1 and primer extension analyses suggest that the observed tissue differences in the expression of these mRNAs are due to transcription from multiple initiation sites, whose strength is tissue-dependent. In the 3' untranslated region, comparison of the nucleotide sequence of the rat beta subunit cDNAs to those from other species reveals regions of high sequence similarity.

Animals↗
PubMed1987

Multiple genes encode the human Na+,K+-ATPase catalytic subunit.

A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms.

Amino Acid Sequence↗
PubMed1987

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.

Animals↗
PubMed1987

Binding of nuclear factors to an upstream region of the human A gamma globin gene.

Two regions in the 5' flanking sequence of the human A gamma globin gene have been identified which bind nuclear factors. One of these regions is located between nucleotides -299 and -140 while the other lies between nucleotides -140 and -60. The former sequence is of interest as this region contains several mutations associated with non-deletion HPFHs. It is possible that factors responsible for the formation of these complexes are involved in developmental regulation of this gene. At the present time it is unknown whether this protein binds at or near the site of the mutations associated with HPFH. It is possible that a protein normally binds to this region in adult erythroid cells and represses expression of the gamma globin genes. When this sequence is altered the factor can not bind and expression of gamma globin genes in adult cells occurs. This could explain the continued production of fetal hemoglobin into adult life. In this model the sites of DNA-factor interaction include the nucleotides at -202, -198, -196 etc. such that nucleotide changes at these positions directly affect factor binding. Alternatively, the sites of DNA factor interaction may be sufficiently proximal to the -202 and -196 region such that single base change at -202, -198, and -196 cause perturbations in the DNA that indirectly effect the DNA-factor interaction. Careful footprinting must be carried out to define the exact binding site. The assay described here provides the basis for the purification of this factor and a study of its interaction with DNA.

Base Sequence↗
PubMed1986

Molecular cloning of the rat stomach (H+ + K+)-ATPase.

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.

Adenosine Triphosphatases↗