Subunit cDNA Research Articles

Subunit C of the vacuolar-type ATPase from the vanadium-rich ascidian Ascidia sydneiensis samea rescued the pH sensitivity of yeast vma5 mutants.

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H(+)-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A(1), neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells.

Cloning and expression of hamster telomerase catalytic subunit cDNA.

Topical application of 7,12-dimethylbenz(alpha)-anthracene induces tumors in the hamster cheek pouch. Telomerase activity is increased in the cancer tissues if compared to normal adjacent tissues in both human and hamster oral cancer. In order to achieve a probe and to investigate the putative role of telomerase in oral carcinogenesis using the hamster cheek pouch model, we have cloned the cDNA encoding the hamster telomerase catalytic subunit (hamTERT). The hamster TERT cDNA encoded 1128 amino acids and shared 64% amino acid identity with human TERT and 80% with murine TERT. As noted with human TERT which express several alternatively spliced mRNAs, we have also detected one alternatively spliced hamTERT mRNA in hamster cancer cells. Transient transfection of hamTERT cDNA in a retroviral expression vector reconstituted telomerase activity in the telomerase negative human lung fibroblast IMR90 cells.

Divergence of volatile anesthetic effects in inhibitory neurotransmitter receptors.

The mechanism of volatile anesthetic (VA) action is unknown. Inhibitory receptors for the neurotransmitters gamma-aminobutyric acid (GABA) or glycine are typically positively modulated by VAs and may be important targets for their action. The existence of a GABA receptor subtype (p), which is uniquely inhibited by VAs, suggested a chimeric receptor approach to identify portions of these proteins that may be necessary for anesthetic effects. A silent mutation resulting in the addition of a unique restriction enzyme recognition site was introduced in GABA receptor type A alpha2, glycine alpha1, and p subunit cDNAs. Chimeras were constructed by rejoining restriction digest fragments and were expressed in Xenopus oocytes. Modulation of submaximal agonist-evoked peak currents by the VAs chloroform, enflurane, halothane, or isoflurane was measured using two-electrode voltage clamp. Four chimeras were constructed and designated glyrho, rhogly, alpha2rho, and rhoalpha2. Glyrho formed glycine-gated receptors with currents that were enhanced by chloroform or halothane but were inhibited by enflurane or isoflurane. Chimeras rhogly and rhoalpha2 each formed GABA-gated receptors with currents that were inhibited by chloroform or halothane but enhanced by enflurane or isoflurane. These data show, for the first time, functional divergence of VA action on a single protein target. The VAs in this study fall into two distinct groups with respect to their effects on these receptors. This grouping parallels the chemistry of these compounds. Our results support the involvement of multiple protein domains in the mechanism of VA modulation of GABA and glycine receptors.

Long sleep and short sleep mice differ in nicotine-stimulated 86Rb+ efflux and alpha4 nicotinic receptor subunit cDNA sequence.

In a recent study, we reported that a restriction fragment length polymorphism associated with the alpha4 nicotinic receptor gene (Chrna4) may play a role in regulating differential sensitivity of LS and SS mouse lines to the seizure-inducing effects of nicotine. Since the alpha4 subunit (CHRNA4) is often found as a heteromer with the beta2 subunit (CHRNB2), alpha4 and beta2 cDNAs from the LS and SS mice were cloned and sequenced. A polymorphism in the coding portion of the alpha4 gene was found (1587A to G) which should result in a threonine/alanine substitution at position 529 (T529A). The LS and SS beta2 nicotinic receptor subunit cDNAs were identical. The potential consequences of the alpha4 polymorphism were evaluated using an ion (86Rb+) flux assay that likely measures the function of alpha4beta2-type receptors. LS-SS differences in maximal nicotine-stimulated ion flux were seen when bovine serum albumin (BSA) was not included but this difference was not seen when BSA was included in the perfusion buffer. Current evidence suggests that BSA may alter the ratio of nicotinic receptors that are in the ground state and desensitized forms. Thus, it may be that the Chrna4 T529A substitution leads to a difference in the ratio of the two receptor forms which then promotes differences in receptor function, as well as differential behavioural sensitivity to nicotine.

Differential distribution of the G protein γ3 subunit in the developing zebrafish nervous system

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein α subunit was the sole regulator of intracellular molecules. The G protein βγ complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein γ subunit. The 666 nucleotides of the zebrafish G protein γ subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse γ subunits. BLAST search analysis of GenBank revealed that the zebrafish γ subunit is 93% identical and 97% similar to the mammalian γ3 subunit. The γ3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected γ3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish γ3 subunit to form a signaling heterodimeric complex with a β subunit was tested using a human β2 subunit. The γ3 formed a heterodimer with β2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the β2γ3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a βγ heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluClβ, Hcgbr-2A and Hcgbr-2B) with high amino-acid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluClα, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluClα1, α2 or α3/GBR-2 subunits (∼55% amino-acid identity). The HcGluClα cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluClβ subunits failed to bind [ 3H]ivermectin. The Hcgbr-2B and HcGluClα subunits bound [ 3H]ivermectin with high affinity; the K d values were 70±16 and 26±12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [ 3H] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [ 3H]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70±7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary.

Cloning and expression of a ferritin subunit for Galleria mellonella.

Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.

Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia

Focal cortical dysplasia is characterized by disorganized cortical lamination, dysplastic and heterotopic neurons, and an association with epilepsy. The contribution that dysplastic and heterotopic neurons make to epileptogenesis in focal cortical dysplasia is unknown and the phenotype of these cells may be distinct. The authors hypothesized that the expression of genes encoding glutamatergic (glutamate [GluR] and N-methyl-D-aspartate NMDA receptors [NR]) and gamma-aminobutyric acid A receptor (GABA(A)R) subunits is distinct in dysplastic and heterotopic neurons and that changes in receptor gene expression could be defined in a cell-specific pattern. Single immunohistochemically labeled dysplastic and heterotopic neurons were microdissected from human focal cortical dysplasia specimens obtained during epilepsy surgery. Pyramidal neurons were microdissected from postmortem control cortex and from temporal cortex without dysplasia resected during temporal lobectomy. Poly (A) messenger RNA (mRNA) from single neurons was amplified, radiolabeled, and used to probe complementary DNA (cDNA) arrays containing GluR(1-6), NR(1A,1B), NR(2A-D), and GABA(A)Ralpha(1-6), and -Rbeta(1-3) subunit cDNAS: The relative hybridization intensities of each mRNA-cDNA hybrid were quantified by phosphorimaging. GluR, NR, and GABA(A)R subunit mRNA expression did not differ between control neurons and nondysplastic epilepsy specimens. Expression of GluR(4), NR(2B), and NR(2C) subunit mRNA was increased, and NR(2A) and GABA(A)Rbeta(1) subunit mRNA was decreased in dysplastic compared with pyramidal and heterotopic neurons. In contrast, GABA(A)Ralpha(1), -Ralpha(2), and -Rbeta(2) as well as GluR(1) mRNA levels were reduced in both dysplastic and heterotopic neurons. Differential expression of GluR, NR, and GABA(A)R mRNA in dysplastic and heterotopic neurons demonstrates cell specific gene transcription changes in focal cortical dysplasia. These results suggest that dysplastic and heterotopic neurons may be pharmacologically distinct and make differential contributions epileptogenesis in focal cortical dysplasia.

Beta-Subunit 102-104 residues are crucial to confer FSH activity to equine LH/CG but are not sufficient to confer FSH activity to human CG.

Horse LH/CG (eLH/CG) and donkey LH/CG (dkLH/CG) are strictly LH-specific in their respective homologous species. However, both bind to the FSH receptors from non-equid species, whereas the zebra hormone (zbLH/CG) does not. The FSH/LH ratio of eLH/CG and of the alphadkbetae hybrid is about tenfold higher than that of dkLH/CG and of the alphaebetadk hybrid, showing that the betae subunit contains the structural features responsible for the high FSH activity of eLH/CG. Only six amino acid positions (51, 94, 95, 102, 103 and 106) are unique to the betae subunit when compared with the betadk and betazb subunits. The Gly-Pro and Val-Phe sequences in positions 102-103 of betadk and betae respectively were swapped by site-directed mutations and the mutated beta-subunits cDNAs were cotransfected in COS cells with either alphae or alphadk subunit cDNA. Other mutations were also introduced in 102-103 dkLH/CG beta-subunit: Ala-Ala, Gly-Ala or Ala-Pro. These mutations with Ala-Ala, Gly-Ala or Ala-Pro in the 102-103 betadkLH/CG subunit did not change the FSH/LH ratio of dkLH/CG but the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation promoted a marked increase in the FSH/LH activity ratio. This was observed with the two heterodimers containing alphae or alphadk. Conversely, the Val(102)-Phe(103) mutation in betae led to a dramatic drop in FSH/LH activity ratio of eLH/CG, to a level similar to that of dkLH/CG. Since all FSHs possess a Gly residue at position 104, we introduced the Gly(102)-Pro(103)-Arg(104)-->Val(102)-Phe(103)-Gly(104) mutation in betadk with the expectation that the increase in FSH activity observed with the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation could be potentiated. In fact, the additional Arg(104)-->Gly(104) mutation was found to abolish the increase in FSH activity observed with Gly(102)-Pro(103)-->Val(102)-Phe(103). Mutations Gly(102)-Pro(103)-->Val(102)-Arg(103) or Gly(102)-Pro(103)-Lys(104)--> Val(102)-Arg(103)-Gly(104) were also introduced in human CGbeta (hCGbeta) to compare the impact of these amino acid changes in the well-studied gonadotrophin hCG. The betahCG mutants obtained, co-expressed either with the human or the horse alpha-subunit, did not display any FSH activity. In conclusion, the 102-104 sequence in eLH/CG beta-subunits appears to be of utmost importance for their binding to FSH receptors. However, these results obtained with equid beta-subunits are not transposable to other gonadotrophins as similar mutations in hCGbeta did not lead to any increase in FSH activity.

Investigation of Subunit Function in ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (α2β2). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg2+, which were similar to the WT enzyme. The Km for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.

Characterisation of a cDNA encoding chick eukaryotic translation initiation factor-2 beta.

A full length cDNA for the beta subunit of chick (Gallus gallus) eukaryotic translation initiation factor-2 is described. This cDNA was isolated by screening a chick cDNA library with a probe derived via differential display of developing chick heart tissue. Up-regulated expression of eIF-2 beta mRNA was confirmed by reverse Northern dot blot analysis. eIF-2 beta, together with eIF-2 alpha and eIF-2 gamma, comprise subunits of a complex that promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation. The nucleotide sequence of the chick eIF-2 beta cDNA predicts a protein of 334 amino acids that has 95%, 93%, 56% and 37% sequence identity with rabbit, human, drosophila and yeast eIF-2 beta, respectively. The deduced eIF-2 beta protein contains a number of functional motifs and domains consistent with the putative function of this protein; these include a potential C2-C2 zinc-finger binding domain, three polylysine regions, and three acidic regions.

Cloning and sequence analysis of the basidiomycete Lentinus edodes ribonucleotide reductase small subunit cDNA and expression of a corresponding gene in L. edodes

We have previously isolated the uck1 gene encoding UMP-CMP kinase from the basidiomycete Lentinus edodes (Kaneko et al., 1998). It was shown to be most actively transcribed in hymenophores of mature fruiting bodies of L. edodes. The reduction of NDPs produced by the nucleoside monophosphate kinase to dNDPs has been known to be catalyzed by ribonucleotide reductase (RNR) which consists of a heterodimer of large and small subunits. So we attempted to isolate the L. edodes cDNA(s) of RNR and study the expression in L. edodes of the corresponding gene(s), resulting in an isolation of the small subunit cDNA from a mature fruiting-body cDNA library of the fungus. This cDNA, named Le.rnr2c, was shown to encode a 418 amino acids (aa) protein, named Le.RNR2, of which the deduced aa sequence shows an overall identity of 71.9% to that of Schizosaccharomyces pombe RNR small subunit. The Le.rnr2 gene was found to be most actively transcribed in hymenophores of mature fruiting body of L. edodes. The in situ RNA-RNA hybridization analysis showed the presence of markedly large amount of the Le.rnr2 transcript in both hymenium and outer region of trama in the hymenophore. The same experiment was done for the uck1 gene, obtaining a similar result. The hymenium contains many basidia in which fusion of two nuclei, meiosis, replication, etc. essential for production of basidiospores occur. The outer region of trama is the region branching out into subhymenium. These imply that Le.rnr2 gene (and uck1 gene) play a role mainly in the nucleotide biosynthesis essential both for production of basidiospores and for divergence of trama cells into subhymenium cells in the hymenophore.

Molecular cloning and sequence analyses of calcium/calmodulin-dependent protein kinase II from fetal and adult human brain. Sequence analyses of human brain calciuum/calmodulin-dependent protein kinase II.

The aims of this study were to characterize specific mRNAs and the expression pattern for isoforms of calcium/calmodulin-dependent protein kinase II (CaMKII) in the human brain. We cloned and sequenced the CaMKII alpha and beta subunit cDNAs, and used them to study the CaMKII expression in human brain. Four distinct isoforms of CAMKII were isolated. Two of them were characterized as CaMKII alpha and beta subunits. The other two showed similar nucleotide sequences, but one had a 33-bp insertion relative to the alpha subunit, and the other had a 75-bp deletion relative to the beta subunit. These alterations are located within the variable regions. These two isoforms were characterized as CaMKII alphaB and beta(e). Northern blot analysis showed that a 4.4-kb messenger RNA for the alpha isoform and a 3.9-kb messenger RNA for the beta isoform were expressed in both human fetal and adult brain to different degrees. The results indicate that CaMKII expression is developmentally regulated. The CaMKII isoform expression was confirmed in human fetal and adult brain using RT-PCR with specific primers, which flanked the CaMKII variable regions. The CaMKII alpha, alphaB, beta, beta' and beta(e) isoforms were characterized in both human fetal and adult brain.

Partial male sterility induced in tobacco by overproduction of mRNA of sweet potato small subunit ADP-glucose pyrophosphorylase

Summary Previously we generated transgenic tobacco plants overexpressing sweet potato ADP-glucose pyrophosphorylase (AGPase) small subunit cDNA under control of CaMV 35S promoter. In order to understand the role of small subunit AGPase in pollen development, these transgenic tobacco plants were studied. Nine out of ten transgenic tobacco plants produced more than 50 percnt; aborted pollen grains. The two transgenic lines T4 and T27 produced more than 75 percnt; aborted pollen grains. Analysis of inheritance of the transgene inserts in T4 and T27 revealed that transgene co-segregated with the pollen abortion phenotype. Microscopic examination of aborted pollen grains showed that their cytoplasm did not contain starch granules, nuclei or any other organelles. Transcript levels of the transgene were very high in stamen tissues of transgenic plants. Transcript levels were correlated with the severity of the pollen abortion phenotype in transgenic plants, indicating that the pollen abortion phenotype probably resulted from overproduction of sweet potato small subunit AGPase mRNAs. Western blot analysis revealed that AGPase small subunit polypeptide levels were significantly reduced in stamen tissues of T4 and T27 compared with control plant. These results suggest that AGPase small subunit gene may play an important role in pollen development.

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.

Human α6 AChR subtypes: subunit composition, assembly, and pharmacological responses

Many nicotinic acetylcholine receptor (AChR) subunits are known to be co-expressed with the α6 subunit in neurons. Because α6β4 AChRs assemble inefficiently and α6β2 AChRs not at all, more complex mixtures of human subunit cDNAs were tested for their abilities to form functional AChRs when expressed in Xenopus oocytes. α6β4β3 AChRs produced the largest and most consistent responses. α6α3β2 AChRs exhibited reduced potency for ACh and increased potency and efficacy for nicotine compared to α3β2 AChRs, but similar resistance to functional inactivation after prolonged exposure to nicotine. α6α4β2 AChRs differed little in potency or efficacy for ACh or nicotine compared to α4β2 AChRs, and had similarly high sensitivity to inactivation by prolonged exposure to nicotine. Co-expression of α6 and β2 cRNAs resulted in large numbers of 3H-epibatidine binding sites in the form of large aggregates but not in functional pentameric AChRs. Co-expression of α6, β2, and α5 resulted in assembly of some functional pentameric AChRs. Chimeras with the large extracellular domain of α6 and the rest from either α3 or α4 efficiently formed functional AChRs. Thus, the extracellular domain of α6 efficiently assembles with β2 to form ACh binding sites, but more C-terminal domains cause difficulties in forming pentameric AChRs. Chimeric α6/α3 and α6/α4 AChRs containing either β2 or β4 subunits were blocked by α-conotoxin MII which had previously been reported to be specific for α3β2 AChRs.

Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7–9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit ofsaccharomyces cerevisiae,schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTβ. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that humanCCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3–24 times of those in other tissues. TheCCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327).The genomic structure of the 5′-end ofCCTβ was also described in detail through comparative analysis.

Modulation of calcium current by recombinant GABA(B) receptors.

Two GABA(B) receptor subunits have been cloned: GABA(B1) and GABA(B2). In this study we investigate the coupling of recombinant GABA(B) receptors to calcium channels in differentiated NG108-15 cells, which exhibit many similarities to neurones but in which functional GABA(B) receptors are normally absent. Transfection of GABA(B1) and GABA(B2) subunit cDNAs enables baclofen-mediated inhibition of different calcium channel subtypes and a component of this modulation is voltage-dependent. When transfected individually, GABA(B2), but not GABA(B1), is able to enhance calcium current inhibition over background levels. Further, an antisense oligodeoxynucleotide to GABA(B1) reduces the average functional response in cells transfected with GABA(B2) alone. Assuming that the functional receptor is heteromeric, this suggests that GABA(B1), but not GABA(B2), is expressed endogenously in NG108-15 cells.

Adenovirus-mediated transfer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats.

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.

Cloning of Bullfrog Thyroid-Stimulating Hormone (TSH) β Subunit cDNA: Expression of TSHβ mRNA during Metamorphosis

A thyroid-stimulating hormone β subunit (TSHβ) cDNA encoding both signal peptide and mature TSHβ molecule was cloned from a cDNA library constructed from total RNA of the bullfrog (Rana catesbeiana) adenohypophysis. The bullfrog TSHβ mRNA was estimated by Northern blot analysis to be approximately 1 kb. The deduced amino acid sequence showed 40–61% homologies with the sequences of TSH β subunits of other vertebrates. Using the cDNA as a probe, we measured changes in mRNA expression in metamorphosing tadpoles of R. catesbeiana. The TSH β subunit mRNA level increased progressively throughout prometamorphic stages, reaching its maximum at the end of prometamorphosis. The maximum level was maintained throughout early and mid climax, declining at late climax. These results, together with previously obtained data on plasma prolactin and pituitary prolactin mRNA levels, as well as thyroid hormone levels, are discussed in relation to metamorphic changes occurring in the bullfrog larvae.