Organization and nucleotide sequence of carp gonadotropin alpha subunit genes.

We have used PCR to amplify and align the sequence of two genes encoding cGTH alpha. Both genes comprise four exons and three introns. The organization of cGTH alpha genes is very similar to that of mammalian GTH alpha genes. However, the cGTH alpha genes only span a region of 1.2 kb which is much smaller than those mammalian GTH alpha genes.     

Isolation and characterization of two coho salmon gonadotropins, GTH I and GTH II

Two gonadotropins, GTH I and GTH II, were isolated from pituitaries of spawning coho salmon (Oncorhynchus kisutch) using sequential extractions with ammonium acetate (pH 9.0) and 40% ethanol, precipitation with 80% ethanol, gel filtration chromatography (Sephadex G-100), anion-exchange chromatography (Mono-Q Sepharose), and gel filtration chromatography (Sephadex G-75). Coho salmon GTH I and GTH II stimulated steroidogenesis in vitro in a similar dose-dependent manner when incubated with either ovaries or testes of prepubertal coho salmon. An in vivo bioassay using coho salmon parr demonstrated that coho salmon GTH I and GTH II did not contain thyrotropic activity. Molecular weights were estimated by gel filtration chromatography to be 43,000 and 39,000 for GTH I and GTH II, respectively. Analysis of coho salmon GTH I and GTH II on reversed-phase high-performance liquid chromatography (rpHPLC) revealed that they consist of alpha and beta subunits with N-terminal amino acid residues of Tyr, Gly (alpha, beta of GTH I) and Tyr,Ser (alpha, beta of GTH II). Coho salmon GTH I-beta and GTH II-beta differed from each other in amino acid composition, N-terminal amino acids (Gly vs. Ser), and molecular weights in SDS-PAGE (19,000 vs. 20,000) and had a high degree of chemical similarity to chum salmon GTH I-beta and GTH II-beta, respectively. Specific rabbit antisera to the beta subunits of coho salmon GTH I and GTH II were generated. The observation of two GTHs with distinctly different chemical characteristics in coho salmon is similar to what has previously been found in chum salmon.     

Tropomyosin from the striated muscles of carp (Cyprinus carpio) and of icefish (Channichthys rhinoceratus).

Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart.     

Pike eel (Muraenesox cinereus) gonadotropin. Amino acid sequences of both alpha and beta subunits

The amino acid sequences of pike eel gonadotropin alpha and beta subunits have been determined by standard sequencing analytical methods. The alpha subunit is composed of 93 amino acid residues while the beta subunit comprises 113 amino acid residues. All the invariant half-cystine residues are in the same positions as those found in other gonadotropins. It is noteworthy that the first, putative glycosylation site (Asn56) found in the alpha subunit of other gonadotropins was replaced by Asp56 in the alpha subunit of pike eel gonadotropin. Similarity analyses indicate that both subunits are structurally more similar to other known fish gonadotropin subunits than to those of the mammalian gonadotropins.     

Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin

An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.     

Primary structure of delta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence

Clones carrying cDNA sequences for the delta subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has indicated that this polypeptide consists of 516 amino acids including a hydrophobic prepeptide of 21 amino acids. The delta subunit of the calf muscle acetylcholine receptor, like the alpha, beta and gamma subunits of the same receptor as well as the alpha and gamma subunits of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of amino acid sequence homology between the calf and Torpedo delta subunits (60%) is comparable to that between the beta subunits (59%) and to that between the gamma subunits (56%), but is lower than that between the alpha subunits of the two species (81%). This suggests that the alpha subunit evolved more slowly than the three other subunits. A dendrogram representing the sequence relatedness among the four subunit precursors of the mammalian and fish acetylcholine receptors has been constructed. Some regions of the delta subunit molecule, including the region containing the putative disulphide bridge and that encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between calf and Torpedo. The relative pattern of regional homology is similar for all four subunit precursors.     

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.     

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Cloning and sequencing of the equine and ovine high-affinity IgE receptor β-and γ-chain cDNA

The high-affinity receptor for IgE is expressed on the surface of mast cells and basophils. It is a transmembrane protein with one alpha, one beta and two gamma subunits. The cDNA sequences for the alpha subunit have already been determined. We report here the cDNA sequences for the beta and gamma subunits. The cytoplasmic domains of these subunits are important for intracellular signalling and the deduced amino acid sequences show the expected immunoreceptor tyrosine-based activation motifs. The gamma subunit is highly conserved between species but more variation is seen with the beta subunit. Near the C terminus of the equine beta chain there is a two-base deletion, which changes the reading frame: residue 237 (human numbering) becomes Asp instead of Glu and the chain is three amino acids shorter than the other known mammalian and rodent sequences.     

Molecular cloning of telomere-binding protein genes from Stylonychia mytilis.

A telomere-binding protein heterodimer of 56 kDa (alpha) and 41 kDa (beta) subunits binds specifically to Oxytricha nova telomeres. Genes encoding both subunits have been cloned previously. Here we report molecular cloning and sequence analysis of the homologous genes in Stylonychia mytilis. The derived amino acid sequences were 79% identical for the alpha subunits and 77% identical for the beta subunits. Three repeats of a Leu/Ile heptad were found in each subunit, which might be involved in heterodimer formation. A 360 amino acid region of the Stylonychia mytilis alpha subunit was found to share weak sequence similarity with human vimentin, suggesting the possibility of a relationship between telomeres and intermediate filaments.     

Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases

The genes encoding proteins responsible for activity of the E1 component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined. Open reading frames encoding E1 alpha (bkdA1, 1233 bp) and E1 beta (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits. The Mr of E1 alpha was 45,158 and of E1 beta was 37,007, both calculated without N-terminal methionine. The deduced amino acid sequences of E1 alpha and E1 beta had no similarity to the published sequences of the E1 subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the E1 alpha subunits of Pseudomonas and rat liver branched-chain-oxoacid dehydrogenases. In particular, the region of the E1 alpha subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1 alpha subunit. There was also considerable similarity between the E1 beta subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.     

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1 beta, high affinity immunoglobulin E Fc receptor gamma subunit and serum amyloid A

Suppression subtractive hybridisation (SSH) is a powerful means to identify genes of cytokines and other genes that express small amount of mRNA. In this study, cDNA of normal fish (carp) head kidney cells (HKC) was subtracted from pooled cDNA of HKC and peritoneal cell (PC) obtained from fish which had been injected with sodium alginate (SA) and scleroglucan (SG) 3-48 h earlier. This subtraction produced 248 clones of cDNA fragments. After sequencing some of the fragments of interest were used as probes, and yielded full-length cDNAs homologous to mammalian interleukin-1 beta (IL-1 beta), the gamma subunit of high affinity Fc receptor for IgE (Fc epsilon RI gamma) and serum amyloid A (SAA); these were cloned and sequenced. Carp IL-1 beta shows 21.8-24.7% amino acid identities to mammalian mature IL-1 beta, and lacks a signal sequence, which is consistent with mammalian IL-1 beta. Carp Fc epsilon RI gamma, which was the first cloned non-mammalian Fc receptor subunit, shows 39.3-40.4% amino acid identities to mammalian Fc epsilon RI gamma, and contains the immunoreceptor tyrosin-based activation motif characteristic of the signal transduction subunit of antigen- and Fc-receptors. Carp SAA is most similar to mammalian acute phase responsive type SAA with 53.0-55.3% amino acid identities. Both SA-elicited and SG-elicited PC expressed higher amounts of IL-1 beta and SAA mRNA compared to saline-injected fish HKC and PC, indicating that these proteins are associated with inflammatory responses, similar to mammalian homologues. Fc epsilon RI gamma was constitutively expressed in leucocytes and not immunopotentiator-responsive, but this indicates that Fc receptor including Fc epsilon RI gamma subunit is likely functional in the carp immune system.     

The voltage-sensitive sodium channel from rabbit skeletal muscle. Chemical characterization of subunits

The alpha and beta subunits of the rabbit skeletal muscle sodium channel have been separated and isolated preparatively under denaturing conditions. In this sodium channel, the beta subunit is not linked covalently to the alpha subunit. The isolated subunits have been subjected to amino acid and carbohydrate analysis. Both subunits are heavily glycosylated (alpha = 26.5%, beta = 29.7% carbohydrate by weight) with N-acetylneuraminic acid and N-acetylhexosamines representing the predominant monosaccharides in each. Enzymatic deglycosylation with neuraminidase and endoglycosidase F yielded single core peptides of approximately 209 kDa for the alpha subunit and 26.5 kDa for the beta subunit. Based on the known carbohydrate composition, the molecular masses for the glycosylated subunits are, therefore, 285 and 37.5 kDa for alpha and beta, respectively. Using the isolated subunits, we calibrated our protein-labeling system on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined the subunit stoichiometry for the rabbit skeletal muscle channel; in the native preparation, the molar ratio of alpha:beta is 1 : 1.     

Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms

Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.     

Identification of an amino acid sequence within GABA(A) receptor beta3 subunits that is important for receptor assembly

GABA(A) receptors are chloride ion channels that can be opened by GABA, the most important inhibitory transmitter in the CNS. In the mammalian brain the majority of these pentameric receptors is composed of two alpha, two beta and one gamma subunit. To achieve the correct order of subunits around the pore, each subunit must form specific contacts via its plus (+) and minus (-) side. To identify a sequence on the beta3 subunit important for assembly, we generated various full-length or truncated chimeric beta3 constructs and investigated their ability to assemble with alpha1 and gamma2 subunits. It was demonstrated that replacement of the sequence beta3(76-89) by the homologous alpha1 sequence impaired assembly with alpha1 but not with gamma2 subunits in alpha1beta3gamma2-GABA(A) receptors. Other experiments indicated that assembly was impaired via the beta3(-) side of the chimeric subunit. Within the sequence beta3(76-89) the sequence beta3(85-89) seemed to be of primary importance for assembly with alpha1 subunits. A comparison with the structure of the acetylcholine-binding protein supports the conclusion that the sequence beta3(85-89) is located at the beta3(-) side and indicates that it contains amino acid residues that might directly interact with the (+) side of the neighbouring alpha1 subunit.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.