Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain

Screening of a rat brain cDNA library with a radiolabeled probe made from an alpha 3 cDNA (Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemanns, S., and Patrick, J. (1986) Nature 319, 368-374) resulted in the isolation of a clone whose sequence encodes a protein, beta 3, which is homologous (40-55% amino acid sequence identity) to previously described neuronal nicotinic acetylcholine receptor subunits. The encoded protein has structural features found in other nicotinic acetylcholine receptor (nAChR) subunits. Two cysteine residues that correspond to cysteins 128 and 142 of the Torpedo nAChR alpha subunit are present in beta 3. Absent from beta 3 are 2 adjacent cysteine residues that correspond to cysteines 192 and 193 of the Torpedo subunit. In situ hybridization histochemistry, performed using probes derived from beta 3 cDNAs, demonstrated that the beta 3 gene is expressed in the brain. Thus, beta 3 is the fifth member of the nAChR gene family that is expressed in the brain. The pattern of beta 3 gene expression partially overlaps with that of the neuronal nAChR subunit genes alpha 3, alpha 4, or beta 2. These results lead us to propose that the beta 3 gene encodes a neuronal nAChR subunit.     

The nicotinic acetylcholine receptor gene family of the pufferfish,Fugu rubripes

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission at nerve-muscle junctions and in the brain. However, the complete gene family of nAChRs has not so far been reported for any vertebrate organism. We have identified the complete nAChR gene family from the reference genome of the pufferfish, Fugu rubripes. It consists of 16 alpha and 12 non-alpha candidate subunits, making it the largest vertebrate nAChR gene family known to date. The gene family includes an unusual set of muscle-like nAChR subunits comprising two alpha1s, two beta1s, one delta, one epsilon, and one gamma. One of the beta1 subunits possesses an aspartate residue and N-glycosylation sites hitherto shown to be necessary for delta-subunit function. Potential Fugu orthologs of neuronal nAChR subunits alpha2-4, alpha6, and beta2-4 have been identified. Interestingly, the Fugu alpha5 counterpart appears to be a non-alpha subunit. Fugu possesses an expanded set of alpha7-9-like subunits and no alpha10 ortholog has been found. Two new candidate beta subtypes, designated beta5 and beta6, may represent subunits yet to be found in the human genome. The Fugu nAChR gene structures are considerably more diverse than those of higher vertebrates, with evidence of "intron gain" in many cases. We show, using RT-PCR, that the Fugu nAChR subunits are expressed in a variety of tissues.     

SB subregion of the human major histocompatibility complex: gene organization, allelic polymorphism and expression in transformed cells

The SB region of the human major histocompatibility complex (MHC) has been cloned from cosmid and lambda phage libraries made from the human B-lymphoblastoid cell line Priess (DR4/4, DC4/4, SB3/4). Two alpha genes and two beta genes are encoded in the 100 kb long SB region in the order SB alpha-SB beta-SX alpha-SX beta. The SB alpha and SB beta genes encode the alpha and beta subunits of the SB subset of class II MHC molecules. Both the SX alpha and the SX beta genes are pseudogenes in the haplotype examined. From the isolated clones, the two haplotypes of the Priess cell line, SB3 and SB4, are distinguished by nucleotide sequencing and blot hybridization analyses. Restriction site polymorphisms between the SB3 and SB4 clones were observed only in relatively small regions of the SB beta and SX beta genes. A mouse macrophage cell line was transfected with one of the cosmid clones containing both SB alpha and SB beta genes. Expression of the alpha and beta genes was detected by fluorescene-activated cell sorting (FACS) and two-dimensional gel electrophoresis using SB-specific monoclonal antibodies.     

Molecular Characterization and Imidacloprid Selectivity of Nicotinic Acetylcholine Receptor Subunits from the Peach-Potato Aphid Myzus persicae

The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicae alpha-subunit cDNAs, Mp alpha1 and Mp alpha2, have been cloned previously. Here we report the isolation of three novel alpha-subunit genes (Mp alpha3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect alpha subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-alpha subunits are clearly different from vertebrate neuronal beta and muscle non-alpha subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR alpha-subunit cDNAs with the rat beta2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the alpha subtype. This is the first demonstration that imidacloprid selectively acts on Mp alpha2 and Mp alpha3 subunits, but not Mp alpha1, in M. persicae.     

Molecular cloning of the cDNA encoding the third polypeptide (gamma) of brain calmodulin-dependent protein kinase II

cDNA clones for three distinct types of rat brain calmodulin-dependent protein kinase II have been isolated. Two of them were identified as cDNA clones for the alpha and beta subunits of this kinase. The other showed a nucleotide sequence similar but, not identical, to that encoding either the alpha or beta subunit. The cDNA sequence encoded a polypeptide, designated gamma, consisting of 527 amino acid residues with a molecular weight of 59,038. The deduced amino acid sequence of gamma was 84 and 87% homologous to those of alpha and beta, respectively. Higher homologies of the sequences were found in the amino-terminal halves of the three species, alpha, beta, and gamma. RNA blot analysis revealed that the mRNAs for alpha, beta, and gamma were expressed in rat brain with different regional specificities.     

Primary structure and gene localization of human prolidase

Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme.     

Alpha-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.     

mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae.

The highly purified yeast mRNA capping enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.     

Cloning and sequencing of the genes of 2-hydoxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the lambda vectors gt11 and EMBL 3 were screened with antisera raised against 2-hydroxyglutaryl-CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a lambda gt11 clone with an insert of 2050 bp and one was a lambda EMBL-3 clone with an insert of approximately 11,000 bp. Escherichia coli cells infected with the lambda gt11 clone expressed the alpha subunit of the dehydratase (Mr, 53,870), whereas with the lambda EMBL-3 clone, the alpha and beta subunits (Mr, 41,857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (alpha) and hgdB (beta) were obtained. The genes have the following order: A-B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the alpha and beta subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in alpha, including a CNC and two CC clusters, and nine in beta) but no similarities to other known protein sequences were found.     

Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

Cloning and sequence analysis of a cDNA for human glycosylasparaginase. A single gene encodes the subunits of this lysosomal amidase

We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the alpha and beta subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (alpha) and 15 (beta) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the alpha and beta subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.