Sequence Of Alpha Subunit Research Articles

Phylogeography of the intertidal barnacle Chthamalus barnesi in the northwestern Indian Ocean

The phylogeography of intertidal invertebrates in the northwestern Indian Ocean is poorly known due to difficulties in crossing diverse political regions for sampling. This study, based on mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear sodium-potassium ATPase alpha-subunit (NaK) sequences, is the first assessment of the phylogeography of the intertidal barnacle Chthamalus barnesi in the Indian Ocean and includes all peripheral seas of the northwestern region. Our phylogenetic analyses defined 1 monophyletic clade, suggesting that C. barnesi does not comprise any cryptic species. Phylogeographic analyses showed no clear differentiation among populations of C. barnesi, except in the Red Sea. This isolated population apparently did not leave the Red Sea during post-Pleistocene glaciation. High ΦST values between the Red Sea and other populations is further indication of isolation. Low values of ΦST among the Persian Gulf, Gulf of Oman and the northeastern Arabian Sea indicate gene flow among these populations. Analysis of molecular variance revealed no significant genetic differentiation among these 3 areas. Phylogeographic analyses suggested that there may be 2 major populations (Red Sea and northwestern Indian Ocean) of C. barnesi in the Indian Ocean resulting from interactions between the Pleistocene glaciation and present-day ocean currents. Among these populations, the Persian Gulf seems to have a higher probability of being the ancestral population. After the Red Sea, the Persian Gulf has the highest genetic diversity of the cox1 gene. This evidence suggests that the Arabian Sea population may have originated from the expansion of the Persian Gulf population after the Pleistocene glaciation.

Planktic cyanobacteria from freshwater localities in Thuathien-Hue province, Vietnam. III. Phylogenetic inference based on partial phycocyanin sequences, morphological and toxicological characters

The morphology, toxicity and partial sequences of beta and alpha subunits including the intergenic spacer (cpcBA-IGS) of the phycocyanin operon were studied in 21 strains of nonaxenic cyanobacterial cultures. The cultures were established from water samples collected in the Thuathien Hue province in Vietnam, 2004. Based on testing for the presence of the mcyA gene, microcystin-production of Microcystis aeruginosa, M. botrys and M. panniformis was confirmed. The presence of the mcyA gene in a strain of Microcystis flos-aquae and a strain of Planktothrix spiroides (HA308) revealed their toxic potential. The phylogeny of the Vietnamese cyanobacteria was inferred using sequences encoding the cpcBA genes. There was no correlation between toxic characters based on ELISA, HPLC and mcyA gene and the phylogeny based on partial cpcBA sequences. Although the resulting tree topology assisted in resolving the phylogeny of some strains of Anabaena, Dolichospermum and Planktothrix, taxonomical problems still exist at the species level for Lyngbya and Microcystis. Future morphological and molecular studies will have to solve these issues.

Haloleptolyngbya alcalis gen. et sp. nov., a new filamentous cyanobacterium from the soda lake Nakuru, Kenya

The food web of the saline-alkaline Lake Nakuru is dominated by the cyanobacterium Arthrospira fusiformis as the primary producer and a huge population of Lesser Flamingos as direct consumers. However, the dense blooms of Arthrospira are not stable, and collapse irregularly and unpredictably. During such periods they are replaced by other algae or cyanobacteria. The wide fluctuation in the cyanobacterial and algal populations of Lake Nakuru has a great influence on food availability for Lesser Flamingos, and is therefore of high ecological importance. To support the descriptive work on these phenomena, we describe here a new cyanobacterial taxon from this soda lake: Haloleptolyngbya alcalis Dadheech, Mahmoud, Kotut et Krienitz gen. et sp. nov. The study was based on multilocus molecular analyses of 16S rRNA gene, 16S-23S internal transcribed spacer, partial sequences of beta and alpha subunits including intergenic spacer (cpcBA-IGS) of phycocyanin operon, phenotypic features using light microscopy, scanning electron microscopy, transmission electron microscopy, and ecology. The new taxon established a separate lineage within the family of Peudanabaenaceae (Oscillatoriales).

Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences

Dadheech P.K., Ballot A., Casper P., Kotut K., Novelo E., Lemma B., Pröschold T. and Krienitz L. 2010. Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences. Phycologia 49: 361–372. DOI: 10.2216/09-71.1Arthrospira comprises multicellular, cylindrical, usually screwlike coiled trichomes and is cultivated commercially. In this study, 33 new strains of Arthrospira isolated from plankton samples collected in Mexico, East Africa and India were investigated and compared with 53 strains or samples of earlier considerations. The study included observations of morphological features and molecular phylogenetic analyses on the basis of nucleotide sequences of internal transcribed spacer (ITS) between 16S rRNA and 23S rRNA genes and partial sequences of beta and alpha subunits including intergenic spacer (cpcBA-IGS) of phycocyanin operon. Morphological traits of Arthrospira such as trichome width, type of coiling and apical cell were not always consistent in culture conditions. It was revealed that Arthrospira phylogeny on the basis of cpcBA-IGS locus was broadly comparable with the ITS region as both phylogenetic trees derived from nucleotide sequences could be divided into two main clusters. Cluster I comprised sequences from American strains mainly, whereas cluster II contained the sequences of the strains originating from Africa and Asia chiefly. Both genetic regions of the strains investigated in the present study coincidently showed a significant sequence divergence among Arthrospira strains from East Africa, India and Mexico indicating possible distinct evolutionary lineages.

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

Uniformity of organellar DNA in Aldrovanda vesiculosa, an endangered aquatic carnivorous species, distributed across four continents

Organellar DNA from the widely distributed but rare and critically endangered aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) was examined. Six chloroplast intergenic regions (3700 nt in total) were sequenced before analyzing the Southern-RFLP (Restriction Fragment Length Polymorphism) of 2 mt gene flanking regions. Only two different chloroplast haplotypes among 15 A. vesiculosa accessions from Africa, Australia, Europe, and Japan were found, generally distinguishing European and non-European plants, with two exceptions. Genetic variation observed in A. vesiculosa appears to be even lower than in other aquatic species with a similar world-wide distribution. A recent bottleneck followed by long-distance dispersal by water birds or low mutation rates could be responsible for the observed genetic uniformity. Estimation of genetic distances based on six chloroplast intergenic regions led to the conclusion that the chloroplast genome of A. vesiculosa matches more closely to that of Drosera regia than Dionaea muscipula, a sister genus sharing snapping traps. The inconsistency between genetic distance estimates based on nuclear and cytoplasmic markers may reflect a chloroplast capture. In A. vesiculosa, a four amino acids substitution (TGWS) in the amino acid sequence of ATP synthase alpha subunit (ATP1), highly conserved mitochondrial protein, was discovered, unique among all organisms based on current knowledge.

Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation

In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available. Coal-tar-contaminated soil was collected, which consisted of 122.5 mg g(-1) total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (alpha-subunit) common to all PAH dioxygenase enzymes and (iii) beta-subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha-, Epsilon- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of alpha-subunit and beta-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe-2S] cluster binding site suggested that these gene fragments encode for alpha-subunit of dioxygenase gene. Sequencing of the cloned libraries representing alpha-subunit gene fragments (Rf1) and beta-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil. The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.

High light induced accumulation of two isoforms of the CF1 alpha-subunit in mesophyll and bundle sheath chloroplasts of C4 plants.

The effect of light irradiance on the amount of ATP synthase alpha-subunit in mesophyll (M) and bundle sheath (BS) chloroplasts of C(4) species such as maize (Zea mays L., type NADP-ME), millet (Panicum miliaceum, type NAD-ME) and guinea grass (Panicum maximum, type PEP-CK) was investigated in plants grown under high, moderate and low light intensities equal to 800, 350 and 50 micromol photons m(-2) s(-1), respectively. The results demonstrate that alpha-subunit of ATP synthase in both M and BS chloroplasts is altered by light intensity, but differently in the investigated species. Moreover, we identified two isoforms of the CF(1) alpha-subunit, called alpha and alpha. The CF(1) alpha-subunit was the major isoform and was present in all light conditions, whereas alpha was the minor isoform in low light. A strong increase in the level of the alpha-subunit in maize mesophyll and bundle sheath thylakoids was observed after 50 h of high light treatment. The alpha and alpha-subunits from investigated C(4) species displayed apparent molecular masses of 64 and 67 kDa, respectively, on SDS/PAGE. The presence of the alpha-subunit of ATPase was confirmed in isolated CF(1) complex, where it was recognized by antisera to the alpha-subunit. The N-terminal sequence of alpha-subunit is nearly identical to that of alpha. Our results indicate that both isoforms coexist in M and BS chloroplasts during plant growth at all irradiances. We suggest the existence in M and BS chloroplasts of C(4) plants of a mechanism(s) regulating the ATPase composition in response to light irradiance. Accumulation of the alpha isoform may have a protective role under high light stress against over protonation of the thylakoid lumen and photooxidative damage of PSII.

Cloning of the Elk Common Glycoprotein .ALPHA.-subunit and the FSH and LH .BETA.-subunit cDNAs

We report the nucleotide and deduced amino acid sequences of the pituitary glycoprotein hormone common alpha-subunit, and the FSH and LH beta-subunits of the elk (Cervus elaphus). These sequences were obtained by RT-PCR of pituitary gland polyadenylated RNA. A partial genomic fragment of the elk follicle stimulating hormone beta-subunit was also amplified from genomic DNA and sequenced. These sequences show high similarity to other related domestic species, including sika deer, ovine, bovine, porcine and equine. The presently reported cDNAs were used as probes for Northern analysis of pituitary gland and muscle samples from elk and sheep. Mature mRNA transcripts of approximately 700, 1700 and 550 bases were expressed in the pituitary gland but not in muscle tissue, for the glycoprotein hormone common alpha-subunit and FSH and LH beta-subunits, respectively. These observations are consistent with reports for other related species. The genomic fragment of elk FSH beta-subunit revealed an intronic microsatellite of 6 CT repeats, which differs from the 19 CT repeats documented for the sheep genomic sequence. We propose that differences in the CT repeats of the elk FSH beta-subunit gene may be used as a marker for elk parentage testing. The elk DNA sequences presently reported represent a unique addition to the growing collection of the pituitary hormone genes for various vertebrates, which will facilitate evolutionary and phylogenetic studies of the gonadotropin genes. In addition, this information and the cloned cDNAs will be useful for studying seasonal expression of the elk pituitary glycoprotein genes.

A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy.

We describe a novel signaling mechanism mediated by the G-protein-coupled receptor (GPCR) angiotensin II (Ang II) type 2 receptor (AT(2)). Yeast two-hybrid studies and affinity column binding assay show that the isolated AT(2) C-terminus binds to the transcription factor promyelocytic zinc finger protein (PLZF). Cellular studies employing confocal microscopy show that Ang II stimulation induces cytosolic PLZF to co-localize with AT(2) at the plasma membrane, then drives AT(2) and PLZF to internalize. PLZF slowly emerges in the nucleus whereas AT(2) accumulates in the perinuclear region. Nuclear PLZF binds to a consensus sequence of the phosphatidylinositol-3 kinase p85 alpha subunit (p85 alpha PI3K) gene. AT(2) enhances expression of p85 alpha PI3K followed by enhanced p70(S6) kinase, essential to protein synthesis. An inactive mutant of PLZF abolishes this effect. PLZF is expressed robustly in the heart in contrast to many other tissues. This cardiac selective pathway involving AT(2), PLZF and p85 alpha PI3K may explain the absence of a cardiac hypertrophic response in AT(2) gene-deleted mice.

ω-Conotoxin CVID Inhibits a Pharmacologically Distinct Voltage-sensitive Calcium Channel Associated with Transmitter Release from Preganglionic Nerve Terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.

SRPDB: Signal Recognition Particle Database.

The Signal Recognition Particle Database (SRPDB) at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html and http://bio.lundberg.gu.se/dbs/SRPDB/SRPDB.html assists in the better understanding of the structure and function of the signal recognition particle (SRP), a ribonucleoprotein complex that recognizes signal sequences as they emerge from the ribosome. SRPDB provides alphabetically and phylogenetically ordered lists of SRP RNA and SRP protein sequences. The SRP RNA alignment emphasizes base pairs supported by comparative sequence analysis to derive accurate SRP RNA secondary structures for each species. This release includes a total of 181 SRP RNA sequences, 7 protein SRP9, 11 SRP14, 31 SRP19, 113 SRP54 (Ffh), 9 SRP68 and 12 SRP72 sequences. There are 44 new sequences of the SRP receptor alpha subunit and its FtsY homolog (a total of 99 entries). Additional data are provided for polypeptides with established or potential roles in SRP-mediated protein targeting, such as the beta subunit of SRP receptor, Flhf, Hbsu and cpSRP43. Also available are motifs for the identification of new SRP RNA sequences, 2D representations, three-dimensional models in PDB format, and links to the high-resolution structures of several SRP components. New to this version of SRPDB is the introduction of a relational database system and a SRP RNA prediction server (SRP-Scan) which allows the identification of SRP RNAs within genome sequences and also generates secondary structure diagrams.

Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci.

1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.

Antigen 43, the major phase-variable protein of the Escherichia coli outer membrane, can exist as a family of proteins encoded by multiple alleles.

agn43 encodes a major phase-variable outer-membrane protein, antigen 43 (Ag43), involved in autoaggregation of Escherichia coli cells. The gene is present in single copy on the chromosome of E. coli K-12. In contrast, Southern hybridization and gene inactivation studies demonstrate that control producer strain E. coli ML308-225 possesses duplicate copies of agn43 (agn43A and agn43B). Construction and analyses of single and double knockout mutants clearly show that both alleles are capable of expressing antigen in a phase-variable manner, with observed differences in the ON<-->OFF switch frequencies appearing to favour expression of Ag43B under conditions of normal laboratory growth. Comparative analysis of agn43A and agn43B gene sequences revealed 98% identity at the nucleotide and predicted protein levels, with differences in the protein sequence of the surface-expressed alpha(43) subunit altering the surface probability of one of the predicted epitopes. Analysis of a panel of enteropathogenic E. coli strains by Southern hybridization using agn43-specific gene probes provided strong evidence for the presence of varying numbers of agn43 alleles within clinical isolates. Taken together, the results indicate the presence of a family of distinct Ag43 proteins encoded by multiple chromosomal alleles.

Phylogenomic analysis of the alpha proteasome gene family from early-diverging eukaryotes.

We employed a phylogenomic approach to study the evolution of alpha subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven alpha proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple alpha subunit sequences generated single alpha proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct alpha proteasome genes each. The presence of seven distinct alpha proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the alpha proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the alpha proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented.

Structural basis for the activation of 20S proteasomes by 11S regulators.

Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.

Male-biased mutation rates revealed from Z and W chromosome-linked ATP synthase alpha-subunit (ATP5A1) sequences in birds.

Whether the mutation rate differs between sexes has been a matter of discussion for years. Molecular analyses of mammals have indicated that males mutate more often than females, as manifested by the faster rate of neutral sequence evolution on the Y chromosome than on the X chromosome. However, these observations can as well be interpreted as specific reduction of the X chromosome mutation rate, which would be adaptive because of reducing the number of slightly deleterious recessive mutations exposed in hemizygote males. Recently, data from birds have suggested that vertebrate mutation rates may indeed be male-biased. In birds, females are the heterogametic sex (ZW), and analyses of the Z-linked CHD1Z gene have shown that it evolves faster than its W-linked and thus female-specific homologue, CHD1W. We have now studied the second avian gene known to exist in a copy on the nonrecombining regions of both the Z and the W chromosome, viz., the ATP synthase alpha-subunit (ATP5A1). In independent comparisons of three pairs of bird species from divergent lineages, intron sequences of the Z-linked copy (ATP5A1Z) were consistently found to evolve faster than the W-linked copy (ATP5A1W). From these data we calculated male-to-female mutation rate ratios (alpha) of 1.8, 2.3, and 5.0 in Galliform, Anseriform, and Ciconiiform lineages, respectively. Therefore, this study provides independent support for a male-biased mutation rate in birds.

SRPDB (signal recognition particle database).

The signal recognition particle database (SRPDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and organizes SRP-related information about SRP RNA, SRP proteins and the SRP receptor. SRPDB is accessible on the WWW at the URL http://psyche.uthct.edu/dbs/SRPDB/SRPDB.++ +html. A mirror site of the SRPDB is located in Europe at the University of Göteborg, Sweden (http://www.medkem. gu.se/dbs/SRPDB/SRPDB.html ). This release of SRPDB adds 10 new SRP RNA sequences (a total of 117 SRP RNAs), four protein SRP19 sequences (a total of 15), seven new SRP54 (ffh) sequences (a total of 52), and eight sequences of the SRP receptor alpha subunit (FtsY) (total of 36). Sequences are arranged in alphabetical and phylogenetic order and alignments are provided which highlight base paired and conserved regions. SPRDB also provides motifs to find new sequences, a brief introduction to SRP function in protein secretion, numerous SRP RNA secondary structure diagrams, 3-D SRP RNA models, and recently obtained crystal structure PDB coordinates of the human SRP54m domain.

Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation.

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.