The α- and β-Tubulin Genes of Euplotes octocarinatus

ABSTRACT. The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.     

Protein—nucleic acid interactions in bacteriophage φ29 DNA replication

Abstract: φ29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3'–5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

A novel fluorescent α-conotoxin for the study of α7 nicotinic acetylcholine receptors

Homomeric α7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified α9 subunit can also form functional homopentamers as well as α9α10 heteropentamers. Current fluorescent probes for α7 nicotinic ACh receptors are derived from α-bungarotoxin (α-BgTx). However, α-BgTx also binds to α9* and α1* receptors which are coexpressed with α7 in multiple tissues. We used an analog of α-conotoxin ArIB to develop a highly selective fluorescent probe for α7 receptors. This fluorescent α-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked α7 currents in Xenopus laevis oocytes with an IC₅₀ value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated α-BgTx, Cy3-ArIB[V11L;V16A] did not block α9α10 or α1β1δε receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [¹²⁵I]-α-BgTx binding to mouse hippocampal membranes with a K _i value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXα7R1 cells but not KXα3β2R4, KXα3β4R2, or KXα4β2R2 cells. This labeling could be abolished by pre-treatment with α-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for α7 receptors.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects

α4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in ³[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1β and IL-6. Nicotine suppresses IL-1β and IL-6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro-β-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti-inflammatory effects similar to α7 receptor activation.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

DNA polymerase template switching at specific sites on the φ29 genome causes the in vivo accumulation of subgenomic φ29 DNA molecules

The accumulation of subgenomic phage φ29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage φ29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo . Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGgAACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the φ29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the φ29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic φ29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.     

The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.     

Different binding modes of tropeines mediating inhibition and potentiation of α1 glycine receptors

Tropeines are bidirectional modulators of native and recombinant glycine receptors (GlyRs) and promising leads for the development of novel modulatory agents. Tropisetron potentiates and inhibits agonist-triggered GlyR currents at femto- to nanomolar and micromolar concentrations respectively. Here, the potentiating and inhibitory effects of another tropeine, 3α-(3'-methoxy-benzoyloxy)nortropane (MBN) were examined by voltage-clamp electrophysiology at wild type and mutant α1 GlyRs expressed in Xenopus laevis oocytes. Several substitutions around the agonist-binding cavity of the α1 subunit interface (N46C, F63A, N102A, R119K, R131A, E157C, K200A, Y202L and F207A) were found to reduce or eliminate MBN inhibition of glycine activation. In contrast, the binding site mutations Q67A, R119A and S129A which did not affect MBN inhibition abolished the potentiation of chloride currents elicited by low concentrations of the partial agonist taurine following pre-incubation with MBN. Thus, potentiation and inhibition involve distinct binding modes of MBN in the inter-subunit agonist-binding pocket of α1 GlyRs. Homology modelling and molecular dynamics simulations disclosed two distinct docking modes for MBN, which are consistent with the differential effects of individual binding site substitutions on MBN inhibition and potentiation respectively. Together these results suggest that distinct binding modes at adjacent binding sites located within the agonist-binding pocket of the GlyR mediate the bidirectional modulatory effects of tropeines.     

ADRENERGIC α- AND β-RECEPTORS EXPRESSED BY THE SAME CELL TYPE IN PRIMARY CULTURE OF PERINATAL MOUSE BRAIN

Abstract— The β-adrenergic agonist, isoproterenol and the α- and β-adrenergic agonist. NA. raise the intracellular concentration of cyclic AMP in cultures of dissociated perinatal mouse brain. This rise is prevented by a β- but not by an α-adrenergic antagonist. The maximal level of cyclic AMP reached in the presence of isoproterenol is markedly higher than that found after exposure to NA. However, if NA is used along with an α-adrenergic antagonist, cyclic AMP levels as high as those after isoproterenol are measured. Agonists with α-adrenergic activity including NA decrease the response to isoproterenol. The decrease is blocked by α-adrenergic antagonists. From this and additional evidence it is concluded: (1) The increase in the level of cyclic AMP caused by β-adrenergic agonists is due to β-receptor-mediated stimulation of adenylate cyclase; (2) the inhibition of this effect by α-adrenergic agonists is mediated by adrenergic α-receptors; (3) the α- and β-adrenergic receptors are likely to be located on the same cells, probably the most abundant putative glial precursor cells. The simultaneous stimulation of α- and β-adrenergic receptors on the same cell may be of significance in the regulation of the response to NA.     

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC₅₀ values for blocking α4β4 and α3β4, responding to EC₂₀ concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.     

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.