Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E. coli host designed for assaying pZ189 supF mutational specificity.

Summary Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 × 10^–7 per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.     

DNA photolyase in E. coli: effects on UV mutagenesis by plasmids expressing the phr gene

Derivatives of an E. coli plasmid pKY33 are described having specific insertions or deletions that effect or do not effect the phr gene (for DNA photolyase) carried in this plasmid. The various plasmids are tested to determine which cause an inhibition of UV mutagenesis producing glutamine tRNA ochre suppressor mutations. The inhibition is found to require a functional phr gene, which substantiates our earlier report that amplified DNA photolyase interferes specifically with a category of mutagenesis involving targeting by a pyrimidine dimer.     

Urea inhibition of renal (NA+ + K+)ATPase activity is reversed by cAMP

In the present work we studied the modulation of the effect of urea on the renal (Na+ + K+)ATPase by cAMP. We observed that urea inhibits the (NA+ + K+)ATPase activity in a dose-dependent manner, reaching 60% of inhibition at the concentration of 1M. This effect was completely reversed by dibutyryl-cAMP (dBcAMP) at 5 x 10(-4)M. The effect of dBcAMP was mimicked by 50 units of the catalytic subunit of protein kinase A and completely abolished by 5 x 10(-7)M H89, an inhibitor of protein kinase A. Addition of 1M urea decreases basal phosphorylation of the immunoprecipitated (NA+ + K+)ATPase in 50%, with this effect completely reversed by 5 x 10(-4)M dBcAMP. Furthermore, 5 x 10(-4)M dBcAMP by itself induced (NA+ + K+)ATPase phosphorylation. Taken together these data indicate that cAMP could be, in addition to the organic solutes already known, an important physiological modulator of the deleterious effect of urea on enzyme activity.     

Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.     

Altered expression of the H+ ATPase in Streptococcus faecalis membranes

Evidence is presented that expression of the H+ ATPase in S. faecalis is influenced by the extracellular pH and K+ level during growth. Altered expression was detected by assay of F1 ATPase and electrophoretic analysis of membrane proteins. K+-limited growth caused about a 2-fold increase in the F1 ATPase. The effect of growth at pH 6, 7 and 9 was studied. Compared to cells grown at pH 7, growth at pH 6 increased the F1 ATPase about 2-fold while growth at pH 9 reduced the F1 ATPase by nearly 4-fold. The elevated F1 ATPase activity in the pH 6 cells was associated with an increase in the F1 ATPase alpha and beta subunits in the membrane while the decrease in F1 ATPase in the pH 9 cells was associated with a marked loss of the alpha subunit. It is suggested that intracellular protons may act as effectors which regulate expression of the F1F0 gene cluster at the level of translation.     

H+-ATPase activity of Escherichia coli F1F0 is blocked after reaction of dicyclohexylcarbodiimide with a single proteolipid (subunit c) of the F0 complex

Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).     

Promoter-promoter interactions influencing transcription of the yeast mitochondrial gene, Oli 1, coding for ATPase subunit 9. Cis and trans effects

The gene for ATPase subunit 9 of yeast mitochondria (Oli 1) contains two promoter sequences (Op1 and Op2) separated by 78 nucleotides. Both promoters are transcribed in vivo and in vitro though with different efficiency. The upstream promoter (Op1) is 12-15 times stronger than the downstream promoter (Op2), and this difference in promoter activity is partly attributable to the influence of the +2 nucleotide (Biswas, T. K., and Getz, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 270-274). In addition, the presence of the strong promoter (Op1) in close proximity to the weak promoter (Op2) partially inhibits the expression of the latter (Op2). The relative orientation of the two promoters has no influence on these inhibitory effects. When both promoters are present in the same reaction mixture, the strong promoter always competes effectively with the weak promoter for limited RNA polymerase (trans or competition effect). When the two promoters are present in the same plasmid, there is an inhibitory interaction between them that decreases as the distance between the two promoters increases (cis or position effect). Thus, the difference between the activities of a strong and a weak mitochondrial promoter in tandem is a function of two effects, the trans or competition effect and the cis or position-related effect. A model for promoter-promoter interactions is proposed.     

Improvement in K+-limited growth rate associated with expression of the N-terminal fragment of one subunit (KdpA) of the multisubunit Kdp transporter in Escherichia coli

Mutations in any one of three genes, kdpA, -B, or -C, in Escherichia coli abolish the activity of Kdp, a multisubunit K+-ATPase that belongs to the P-type ATPase family of cation transporters. We found in this study that expression in vivo of a 135-amino-acid-long N-terminal fragment (KdpA'), less than one-quarter the length of native KdpA, was able to mediate an improvement in K+-limited growth rates in two different contexts, even in the absence of both KdpC and the ATPase subunit KdpB. The first context was when KdpA' was overexpressed in cells from a heterologous inducible promoter, and the second was when KdpA' was provided with a C-terminally altered extension (following a spontaneous genetic rearrangement). Our results suggest that KdpA' provides an incipient pathway for K+ translocation which can serve to transport K+ into the cells in response to the cytoplasmic membrane potential.     

In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli

The function of the epsilon subunit of the Escherichia coli proton-translocating ATPase has been examined by using a mutant defective in the uncC gene. Strains with a defective uncC gene show a reduction in both growth yield and growth rate that is more severe than for other unc mutants; this deleterious effect is shown to be a result of the ATPase activity of the F1 complex which is missing the epsilon subunit. In addition, the epsilon-deficient F1 is bound less tightly to the membrane. These data suggest that, in vivo, the epsilon subunit is capable of inhibiting the ATPase activity of F1 and also functions in the binding of F1 to F0.     

The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins.

Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68).     

Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F1-beta monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit- mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit- mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GAL1. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective F0 sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.     

Identification of collaborative activities with oxidative phosphorylation in bipolar disorder

Bipolar disorder (BD) is a psychiatric disease considered to polygenic with multiple factors in genetics, each of which is not dominant but collaborative during pathogenic progression. We describe a method that estimates the collaborative contribution to the disease between a certain well-studied pathway and the other candidate pathway using Gene Set Enrichment Analysis (GSEA). We describe a modified GSEA (improved derivation) to identify genes that are significantly and differentially expressed between disease and non-disease states and that are consistently co-expressed with a target pathway which is deeply related to disease etiology. The modified GSEA uses available gene expression data to identify molecular mechanism (ubiquitin-proteasome and inflammatory response) associated with the disease. We believe that this approach could reveal hidden relations between a certain well-studied pathway and the other candidate pathway known in literature.

Abbreviations

ATP5I - ATP synthase H+ transporting mitochondrial F0 complex subunit E, ATP5J - ATP synthase H+ transporting mitochondrial F0 complex subunit F6, BAD - Bcl-2-associated death promoter, BAX - Bcl-2-associated x protein, Bcl-2 - B-cell lymphoma 2, BDNF - brain derived neurotrophic factor, COX5B - Cytochrome c oxidase subunit Vb, COX7A2 - cytochrome c oxidase subunit VIIa polypeptide 2, DLK - dual leucine zipper-bearing kinase, GABA - Gamma aminobutyric acid, IL-8 - Interleukin 8, NDUFA1 - NADH dehydrogenase 1 alpha subcomplex 1, NDUFB2 - NADH dehydrogenase1 beta subcomplex 2, NDUFS4 - NADH dehydrogenase Fe-S protein 4, NGF - nerve growth factor, PPP2R5C - protein phosphatase 2 regulatory subunit B gamma, PSMA3 - proteasome subunit alpha type 3, PSMA7 - proteasome subunit alpha type 7, PSMB1 - proteasome subunit beta type 1, PSMB6 - proteasome subunit beta type 6, PSMB7 - proteasome subunit beta type 7, PSMC2 - proteasome 26S subunit ATPase 2, PSMC5 - proteasome 26S subunit ATPase 5, SLC6A4 - solute carrier family 6 member 4, TNFa - tumor necrosis factor a, UBE2A - ubiquitinconjugating enzyme E2A, UCRC - ubiquinol-cytochrome c reductase complex, UFC1 - ubiquitin-fold modifier conjugating enzyme 1, UQCRQ - ubiquinol-cytochrome c reductase complex III subunit VII, USP14 - ubiquitin specific protease 14.