Positive and Negative Selection in the β-Esterase Gene Cluster of the Drosophila melanogaster Subgroup

We examine the pattern of molecular evolution of the β-esterase gene cluster, including the Est-6 and ψEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For ψEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of ψEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of ψEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the β-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function. [Reviewing Editor: Dr. Martin Kreitman]     

Structural Insights into the Mechanism and Inhibition of the β-Hydroxydecanoyl-Acyl Carrier Protein Dehydratase from Pseudomonas aeruginosa

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.     

Epidemiology of Extended-Spectrum β-Lactamase Producing Escherichia coli in the Stools of Returning Japanese Travelers,and the Risk Factors for Colonization

Objective

Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers.

Methods

Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL) producing Escherichia coli. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing E. coli positive group (18 cases; 26%) and an ESBL-producing E. coli negative group (50 cases; 74%), and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of bla _CTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted.

Results

In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0–75.0, p<0.0001). There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77%) produced CTX-M-15, no ST131 clone was detected.

Conclusion

We must be aware of the possibilities of acquiring ESBL-producing E. coli during travel in order to prevent the spread of these bacteria not only in Japan but globally.     

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ₂) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr²¹⁸, Tyr²²⁴, and Tyr²²⁸) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr²¹⁸ seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

Insights into the metabolism,lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites'

The archaeal phylum ‘Diapherotrites'' was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the ‘Diapherotrites''. The most complete of the SAGs, Candidatus ‘Iainarchaeum andersonii'' (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the ‘Diapherotrites'' have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. ‘Diapherotrites'' 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation.     

Role of β-lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP-II

The β-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 β-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k_on) and dissociation (k_off) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.     

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.     

Structural Insights into the Epimerization of β-1,4-Linked Oligosaccharides Catalyzed by Cellobiose 2-Epimerase,the Sole Enzyme Epimerizing Non-anomeric Hydroxyl Groups of Unmodified Sugars

Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of β1,4-mannobiose to 4-O-β-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the β5-β6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.     

Structural Requirements of the Photoreceptor Phosphodiesterase ��-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its γ-subunit (Pγ), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pγ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pγ78–87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pγ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pγ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63–70 and/or the intact α2 helix of Pγ stabilize binding of C-terminal Pγ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pγ molecule and tested for activation by transducin, we found that the C-terminal region (Pγ63–87) by itself could not be displaced but that transducin could relieve inhibition of certain Pγ truncation mutants. Our results are consistent with two distinct mechanisms of Pγ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pγ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pγ to effectively relieve its inhibitory constraint.     

Insights into the thermodynamics of copper association with amyloid-β, α-synuclein and prion proteins

This review examines recent studies on the thermodynamics of copper association with amyloid-β, α-synuclein and prion protein, with an eye towards using this information to understand the etiology of associated neurodegenerative diseases. A variety of binding affinities and binding sites, which are essential to understand the function and consequence of copper-protein interaction, have been reported for copper to these three neurobiologic systems. This current review reconciles the disparate models presented in the literature.     

Molecular Dynamics Simulations to Gain Insights into the Stability and Morphologies of K3 Oligomers from β2-microglobulin

Abstract

β2-Microglobulin (β2-m) forms amyloid fibrils in patients undergoing long-term hemodialysis. K3 peptide, a Ser20-Lys41 fragment of β2-m, has been known to form fibrils over a wide range of pH and solvent conditions. Recent solid-state NMR has revealed that K3 oligomer adopts a parallel U-shaped β-strand-turn-β-strand motif. In order to investigate the stability and morphologies of K3 oligomers with different sizes (dimer, trimer, and tetrameri and organizations (single and double layers), several all-atom molecular dynamics simulations were conducted at 310 K and pH 2 in water and 2,2,2-trifluoroethanol (TFE). For single-layered organizations, our results show that TFE destabilizes the stacking of K3 peptides due to the fact that TFE weakens the intermolecular hydrophobic interactions of K3 oligomers. In addition, we also identified that the loop region is stabilized by the hydrophobic cluster involving resides Y7, Fll, and I16. Our results further suggest that K3 tetramer is a potential minimal nucleus seed for the formation of K3 protofibrils. For dou-ble-layered organizations in water, our data demonstrate that K3 peptides can form various stable assemblies through different interfacial arrangements, such as NN, NC, and CC, by different driving forces. We further propose that the stacking of different interfaces between two facing β-sheets of K3 peptides could be related to different fibril morphologies, which is in good agreement with the previous experimental results, showing that K3 protofibrils associated to formed mature fibrils with a wide range of diameters from 4 to 15 nm when they were transferred from 20% (v/v) TFE to aqueous solution.     

Inactivation of β-fucosidase by iodine,N-acetylimidazole and tetranitromethane. Evidence for the existence of essential tyrosine residues

β-Fucosidase (β-d-fucoside fucohydrolase, EC 3.2.1.38) isolated from the digestive juice of Achatina balteata is markedly inactivated when the enzyme is preincubated with three different probes, specific for tyrosine residues in the experimental conditions used. An effective protection against inactivation of the enzyme is obtained in the presence of a substrate analogue. These data strongly suggest that β-fucosidase possesses essential tyrosine residues.     

Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with k _cat/K _m values (at 75 °C, pH 5.5) of about 4.1 × 10⁴M⁻¹s⁻¹ and 3.1 × 10⁴M⁻¹s⁻¹ respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998     

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl- Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,and Isolation and Characterization of the Products

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)₂-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)₂-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(6³-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)₂-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)₂-βCDs were identified as 6¹,6²-, 6¹,6³- and 6¹,6⁴-di-O-(6³-O-α-D-mannosyl-maltotriosyl)-βCD.     

Identification of biosynthetic genes for the β-carboline alkaloid kitasetaline and production of the fluorinated derivatives by heterologous expression

Journal of Industrial Microbiology & Biotechnology - β-Carboline alkaloids exhibit a broad spectrum of pharmacological and biological activities and are widely distributed in nature....     

Characterization of the mRNAs for α-, β- and γ-actin

The mRNAs for α-, β- and γ-actin have been characterized with respect to molecular weight and poly(A) content. Polyacrylamide gel electrophoresis under denaturing conditions shows that the mRNA for α-actin (muscle-specific actin) is approximately 4.6 × 10⁵ daltons in size, and that the mRNAs for β- and γ-actin (nonmuscle actins) are much larger, approximately 6.6 × 10⁵ daltons in size. We therefore calculate that the noncoding regions of the β- and γ-actin mRNAs contain about 800 nucleotides. This is in marked contrast to the noncoding regions of α-actin mRNA which contain only about 180 nucleotides. During electrophoresis in high-resolution nondenaturing gels, the β-actin mRNA migrates slightly slower than the γ-actin mRNA. This indicates either that β-actin mRNA is about 100 nucleotides longer than γ-actin mRNA, or that these mRNAs differ in secondary structure. Fractionation of actin mRNA on the basis of poly(A) content shows that a substantial portion of the β-actin mRNA, but very little of the α- or γ-actin mRNAs, fails to bind to oligo(dT)-cellulose. Much of this poly(A)-deficient β-actin mRNA, however, does bind to poly(U)-Sepharose, a substrate with higher affinity for short poly(A) sequences. This indicates that many of these β-actin mRNA molecules are polyadenylated, but that they have unusually short poly(A) tails. The finding that β- and γ-actins are translated from mRNAs of different electrophoretic mobility and different poly(A) content strongly suggests that these two closely related proteins are products of different genes.     

Modulation of the β-Adrenergic Receptor-Coupled Adenylate Cyclase by Chemical Inducers of Differentiation: Effects on β Receptors and the Inhibitory Regulatory Protein Gi

Abstract

Several drugs known to induce differentiation in tumor cells were analyzed for their effects on the β-adrenergic receptor-coupled adenylate cyclase system in two human carcinoma cell lines, HeLa and A431. Each of the drugs was tested alone or in combination with sodium butyrate (NaBu), a known inducer of this signal transduction system. Puromycine amino nucleoside (PMAN) caused the largest increase in β-adrenergic receptors in HeLa cells followed by hexamethylenebisacetamide (HMBA) whereas 5′-azacytidine (5AZC) was ineffective. In addition, PMAN but not the others acted together with NaBu to elevate receptor levels 12-fold over control values. In contrast, HMBA and 5AZC were much more effective on A431 cells, PMAN caused only a slight increase in β receptors and none of the drugs acted in concert with NaBu. The increase in β receptors was usually accompanied by a corresponding increase in isoproterenol-stimulated adenylate cyclase activity. These effects of the drugs appeared to require protein synthesis as they were blocked by cycloheximide. In addition, some of the drugs caused a substantial decrease in basal adenylate cyclase activity. This effect on basal activity was abolished in cells treated with pertussis toxin, which ADP-ribosylates the inhibitory GTP-binding protein, G_i. Both HeLa and A431 cells contained a 41 kDalton substrate for the toxin which corresponds to the α; subunit of G_i. The G_i subunit was ADP-ribosylated by the toxin to a similar extent in membranes from control and drug-treated cells. Thus, the drugs appear to induce quantitative changes in β-adrenergic receptors and qualitative changes in G_i which results in a highly responsive β-adrenergic-stimulated adenylate cyclase.