Beta Subunit cDNA Research Articles

Cloning, characterization and expression analysis of calcium channel β subunit from pearl oyster (Pinctada fucata)

The absorption, transport and localization of calcium underlie the basis of biomineralization, and Ca(2+) entry into epithelial cell is the primary step in shell formation. However, the related mechanism of Ca(2+) transport is poorly documented at the gene or protein level. L-type voltage-dependent calcium channels may be involved in calcium transport for biomineralization in some marine invertebrates. In this study, a full-length cDNA of a voltage-dependent calcium channel beta subunit from Pinctada fucata (PCabeta) was cloned, and its amino acid sequence was deduced. PCabeta shared 51%-67% apparently sequence identity with voltage-dependent calcium channel beta subunits from other species. However, PCabeta was much shorter than other voltage-dependent calcium channel beta subunits particularly at the carboxyl terminus, indicating that it is likely a truncated beta subunit isoform. Semi-quantitative RT-PCR analysis showed that PCabeta was expressed in all the tested tissues and that it had a higher expression level in the gill tissue and hemolymph than in other tissues, suggesting that L-type voltage-dependent calcium channels are responsible for Ca(2+) absorption in the gill and Ca(2+) entry into hemocytes. In the mantle, PCabeta mRNA was predominantly expressed in the inner and middle folds of the mantle epithelium, suggesting that L-type voltage-dependent calcium channels are involved in Ca(2+) absorption from the ambient medium in the mantle. All these results suggest that voltage-dependent calcium channels are involved in Ca(2+) uptake and transport during oyster biomineralization.

Novel Brugada syndrome‐causing mutation in ion‐conducting pore of cardiac Na+ channel does not affect ion selectivity properties

Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the alpha-subunit of cardiac Na+ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57-year-old male patient. Since the mutation is located in a segment of the ion-conducting pore of the cardiac Na+ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. HEK-293 cells were transfected with wild-type (WT) or D1714G alpha-subunit and beta-subunit cDNA. Whole-cell configuration of the patch-clamp technique was used to study biophysical properties at room temperature (21 degrees C) and physiological temperature (36 degrees C). This study represents the first measurements of human Na+ channel kinetics at 36 degrees C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. D1714G channel yielded nearly 80% reduction of Na+ current density at 21 and 36 degrees C. At both temperatures, no significant changes were observed in V(1/2) values and slope factors for voltage-dependent activation and inactivation. At 36 degrees C, but not at 21 degrees C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient.

Cloning of the Elk TSH .BETA.-Subunit cDNA and Seasonal Expression of the Pituitary Glycoprotein Hormone Genes

We report the elk (Cervus elaphus) thyroid stimulating hormone (TSH) beta-subunit cDNA cloning, nucleotide and deduced amino acid sequences. The TSH beta-subunit cDNA was obtained by RT-PCR of polyadenylated pituitary RNA. The deduced elk TSH beta-subunit peptide chain shares between 93 to 99% sequence similarities with the reported TSH beta-subunit of a sub-set of related species. The TSH beta-subunit gene is expressed in the elk pituitary gland as a mature transcript of approximately 600 bases, which corresponds to the size of the mRNA expressed in the sheep pituitary gland. Seasonal expression of the pituitary gonadotropin genes was investigated by Northern blot analyses. Samples of elk pituitary glands collected during the breeding season showed elevated steady state levels of common alpha-subunit and FSH and LH beta-subunit gene expression, consistent with the seasonal reproductive cycling of this species. Samples collected before the breeding season demonstrated decreased expression of the gonadotropin genes. TSH, which is not directly tied to reproduction, had similar levels of expression, regardless of the animal's reproductive status.

Characterization of two forms of mouse salivary androgen-binding protein (ABP): implications for evolutionary relationships and ligand-binding function.

Mouse salivary androgen-binding protein (ABP) is a member of the secretoglobin family produced in the submaxillary glands of house mice (Mus musculus). We report the cDNA sequences and amino acid sequences of the beta and gamma subunits of ABP from a mouse cDNA library, identifying the two subunits by their pIs and molecular weights. An anomalously high molecular weight of the alpha subunit is likely due to glycosylation at a single site. A phylogenetic comparison of the three subunits of ABP with the chains of other mammalian secretoglobins shows that ABP is most closely related to mouse lachrymal protein and to the major cat allergen Fel dI. An evaluation of the most conserved residues in ABP and the other secretoglobins, in light of structural data reported by others [Callebaut, I., Poupon, A., Bally, R., Demaret, J.-P., Housset, D., Delettre, J., Hossenlopp, P., and Mornon, J.-P. (2000) Ann. N.Y. Acad. Sci. 923, 90-112; Pattabiraman, N., Matthews, J., Ward, K., Mantile-Selvaggi, G., Miele, L., and Mukherjee, A. (2000) Ann. N.Y. Acad. Sci. 923, 113-127], allows us to draw conclusions about the critical residues important in ligand binding by the two different ABP dimers and to assess the importance of ligand binding in the function of the molecule. In addition to the cDNAs, which represent those of the musculus subspecies of Mus musculus, we also report the coding regions of the beta and gamma subunit cDNAs from two other mouse inbred strains which represent the other two subspecies: M. musculus domesticus and M. musculus castaneus. The high nonsynonymous/synonymous substitution rate ratios (K(a)/K(s)) for both the beta and gamma subunits suggest that these two proteins are evolving under strong directional selection, as has been reported for the alpha subunit [Hwang, J., Hofstetter, J., Bonhomme, F., and Karn, R. (1997) J. Hered. 88, 93-97; Karn, R., and Clements, M. (1999) Biochem. Genet. 37, 187-199].

Infertility in mice induced by the rhesus monkey chorionic gonadotropin beta-subunit glycoprotein (rmCGbeta) using DNA immunization.

The recombinant eukaryotic expression vector pCMV4-rmCGbeta, inserted full-length cDNA of the beta-subunit of rhesus monkey chorionic gonadotropin (rmCGbeta), as DNA immuno-contraceptive against CGbeta glycoprotein, has previously demonstrated the biological expression of rmCGbeta in vitro and in vivo. The plasmid DNA of pCMV4-rmCGbeta was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCGbeta DNA delivery method for BALB/c mice, and the dose of 10 microg should be enough to elicit immune response. With different doses from 10-50 microg, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCGbeta DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 microg of pCMV4-rmCGbeta DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCGbeta DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of beta-subunit of mouse chorionic gonadotropin (muCGbeta) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCGbeta shares 99.6% homology with rmCGbeta and 90.6% with hCGbeta respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCGbeta contraceptive should be further studied as a CGbeta DNA contraceptive. (Mol Cell Biochem 231: 89-96, 2002)

Molecular cloning of three nonhuman primate follicle stimulating hormone beta-subunit cDNAs.

The follicle stimulating hormone (FSH) beta-subunit cDNAs were cloned and sequenced for an old world primate, the rhesus monkey (Macaca mulatta), and two New World primates, the common marmoset (Callithrix jacchus) and pygmy marmoset (Cebuella pygmaea). The cDNA and predicted amino acid sequences of the rhesus monkey FSH beta-subunit were related most closely to the human FSH beta-subunit (> 96% identity). The common and pygmy marmosets have identical FSH beta-subunit cDNAs, whereas the marmoset FSH beta-subunit diverges from the rhesus and human molecules with less than 93% identity. These results have significance for the implementation of assisted reproductive technologies in the nonhuman primate as well as the evolution of genes encoding reproductive hormones.

Equine follicle-stimulating hormone: molecular cloning of beta subunit and biological role of the asparagine-linked oligosaccharide at asparagine(56) of alpha subunit.

Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated alpha and beta subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn(56) of the alpha subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH beta subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH alpha56/beta) to investigate the biological role of the oligosaccharide at Asn(56) in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG alpha56/beta) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH alpha56/beta), however, did not show any FSH activity, indicating that the oligosaccharide at Asn(56) was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn(56) of the alpha subunit.

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.

Cloning and regulation of the rat activin betaE subunit.

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

Expression and characterization of recombinant β-subunit hCG homodimer

We have linked two human chorionic gonadotropin (hCG) beta-subunit cDNAs in tandem such that the expressed fusion protein consists of two mature beta-subunits joined through the carboxy terminal peptide of the first beta-subunit. A single glycine residue is inserted between the two subunits in the fusion protein. Chinese hamster ovary (CHO) cells transformed with a clone that contains the fused cDNAs express and secrete a protein that is consistent with it being a beta-hCG homodimer protein. These beta-homodimer molecules can recombine with two free alpha-subunits indicating that both beta-subunits within the homodimer are likely folded in their native conformation. Our data also suggest that the two beta-subunits fold upon each other as a globular protein and do not appear to exist as a simple fusion of two linear beta-subunits. Furthermore, the two beta-monomer subunits in the fusion protein form a stable homodimer that can bind and activate the hLH/CG receptor specifically. Recombination of the fusion protein with alpha-subunits appears to favor an arrangement where two alpha-subunits combine with a single molecule of the fusion protein. The recombined molecule consists of four subunits and is comparable to two tethered hCG moieties, which constitutes a hCG dimer. This hormone dimer can bind and activate the hLH/CG receptor with an activity approximating that of native hCG.

Complementary DNA sequence of chicken thyroid-stimulating hormone (TSH) beta subunit.

Chicken TSH beta subunit cDNA was cloned and sequenced. The cDNA encodes a putative signal peptide and a mature protein consisting of 20 and 114 amino acids, respectively. The amino acid sequence of chicken TSH beta subunit is highly conserved (98.5%) between chicken and Japanese quail, whereas it has a low homology between chicken and mammals (67-69%), an amphibian (58%) and fish (40-49%). Structural characteristics of TSH beta subunits of avian species are discussed in comparison with those of non-avian vertebrates.

Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors.

Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37 degrees C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR beta subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.

Molecular cloning of quail thyroid-stimulating hormone (TSH) beta subunit.

Thyroid-stimulating hormone (TSH) beta subunit cDNA was obtained from Japanese quail (Coturnix japonica) by PCR and its nucleotide sequence was determined. The cDNA encodes a putative signal peptide and a mature protein consisting of 20 and 114 amino acids, respectively. The amino acid sequence of quail TSHbeta subunit shows homologies of 67-69% in mammalian species, 58% in amphibian and 43-49% in teleost fish. Comparison of the amino acid sequence with TSHbeta subunits of other species reveals some differences in several regions responsible for its biological functions and characteristic features of the avian TSHbeta subunit, suggesting that the functional domains have diverged cooperatively between the hormone and its receptor during evolution.

Functional expression of the cDNA encoded by the human ATP1AL1 gene

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (pHi) recovery in Cells loaded with a pH-sensitive dye [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1-encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Comparison of the 86Rb+ influx and proton efflux, however, does not support equal H+/Rb+ exchange mediated by this pump under the conditions of pHi-monitoring experiments. Moreover, whereas the acid extrusion activity mediated by the pump shows a marked pH dependence, the 86Rb+ uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pHi. These data suggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K(+)-dependent ATPase. The possible implications of the discrepancy between 86Rb+ uptake and pHi monitoring data are discussed.

A Novel K+ Channel β-Subunit (hKvβ1.3) Is Produced via Alternative mRNA Splicing

Voltage-gated K+ channels can form multimeric complexes with accessory beta-subunits. We report here a novel K+ channel beta-subunit cloned from human heart, hKv beta 1.3, that has 74-83% overall identity with previously cloned beta-subunits. Comparison of hKv beta 1.3 with the previously cloned hKv beta 3 and rKv beta 1 proteins indicates that the carboxyl-terminal 328 amino acids are identical, while unique variable length amino termini exist. Analysis of human beta-subunit cDNA and genomic nucleotide sequences confirm that these three beta-subunits are alternatively spliced from a common beta-subunit gene. Co-expression of hKv beta 1.3 in Xenopus oocytes with the delayed rectifier hKv1.5 indicated that hKv beta 1.3 has unique functional effects. This novel beta-subunit induced a time-dependent inactivation during membrane voltage steps to positive potentials, induced a 13-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 13 +/- 0.5 ms versus 35 +/- 1.7 ms at -40 mV). Most notably, hKv beta 1.3 converted the Kv1.5 outwardly rectifying current voltage relationship to one showing strong inward rectification. These data suggest that Kv channel current diversity may arise from association with alternatively spliced Kv beta-subunits. A simplified nomenclature for the K+ channel beta-subunit subfamilies is suggested.

Interallelic complementation of beta-subunit defects in fibroblasts of patients with propionyl-CoA carboxylase deficiency microinjected with mutant cDNA constructs.

Propionic acidemia results from deficiency of propionyl-CoA carboxylase (PCC) activity. PCC is a biotin-dependent, mitochondrial enzyme composed of alpha- and beta-subunits (structure, alpha 4 beta 4), with the alpha-subunit containing the biotin ligand. About two-thirds of fibroblast lines from patients with mutations in the PCCB (beta-subunit) gene show interallelic complementation in cell fusion experiments (the pccB and pccC subgroups of the pccBC major group defining beta-subunit mutations, where pccB x pccC fusions show complementation). We previously identified the mutations in several pccB or pccC cell lines and suggested that point mutations or small, in-frame insertions or deletions were likely responsible for the complementation obtained between beta-subunit defects. To test this hypothesis, we have introduced five different mutations (three pccB and two pccC) that fit these criteria into a PCC beta-subunit cDNA plasmid expressed from a cytomegalovirus promoter. The cDNA plasmids were microinjected into mutant fibroblasts and the cells were assayed by radioautographic detection of 14C-propionate incorporation into cellular macromolecules. Four different mutations (Pro228Leu or dupKICK140 from pccB or delta IIe408 or Arg410Trp from pccC) complemented cells from complementation subgroups in a pattern congruent with the results obtained in cell fusion experiments. The fifth mutation, Arg536Asn, which was found both in a complementing pccB and a non-complementing pccBC cell line, failed to complement any of the mutant cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

CDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.

Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.

CDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase

Rab geranylgeranyl transferase (Rab GG transferase) attaches 20-carbon geranylgeranyl groups to cysteine residues in Rab proteins that contain the COOH-terminal sequence Cys-X-Cys or Cys-Cys. Rab GG transferase consists of two components that are separable in high salt solutions. Component A is a 95-kDa protein, and Component B is a heterodimer consisting of a 60-kDa alpha subunit and a 38-kDa beta subunit. In the current paper, we have cloned cDNAs for the alpha and beta subunits of Component B. The cDNAs for the rat alpha and beta subunits predict proteins of 567 and 331 amino acids, respectively. The mRNAs for both subunits are expressed in many tissues. When transfected together in embryonic kidney 293 cells, the alpha and beta subunit cDNAs produced Rab GG transferase activity that was stimulated in vitro by the addition of purified Component A. Comparisons of the amino acid sequences suggest that the proteins encoded by the Saccharomyces cerevisiae genes MAD2 and BET2 are the yeast counterparts of the mammalian Rab GG transferase alpha and beta subunits, respectively.

TSH Beta Subunit Gene Expression in Human Lymphocytes

Production of neuroendocrine peptides by human lymphocytes is thought to facilitate control of the immune response. The presence of neuroendocrine peptide gene expression, specifically the TSH beta subunit gene, was studied in human lymphocytes using Northern blot analysis and polymerase chain reaction (PCR) techniques. Northern blot analysis of human lymphocyte RNA probed with a TSH beta cDNA probe failed to demonstrate TSH beta subunit steady state message levels. PCR amplification of lymphocyte-derived cDNA using TSH beta subunit complementary primers resulted in amplification of a .38 Kb DNA fragment, confirming expression and initial exonic splicing of TSH beta subunit gene exons 2 and 3 in human lymphocytes. Sequence analysis of this .38 Kb DNA fragment demonstrated conservation of exon borders after splicing (exons 2 and 3) and predicted an amino acid translation product similar, if not identical, to human TSH beta peptide sequence. Hybridization with a TSH beta subunit cDNA probe of PCR-amplified reverse-transcribed lymphocyte RNA suggested that: (1) the abundance of TSH beta subunit gene transcripts in human lymphocytes is less than the relative abundance in T3-treated pituitary; and (2) this messenger RNA may be modulated by the presence of certain thyromimetic compounds (T2, T3, TRIAC).