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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 325 records · Page 18Linked to original sources
PubMed1989

Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus.

msDNA is a peculiar molecule consisting of a branched RNA linked to single-stranded DNA via a 2',5' phosphodiester bond. A cell-free system, utilizing cells permeabilized with phenethyl alcohol, was established to study the synthesis of msDNA in M. xanthus. Permeablized cells labeled with [alpha-32P]dCTP in the presence of ddGTP, ddATP, or ddTTP produce a band that migrates at the same position as the full-sized msDNA in an polyacrylamide gel. However, when this band is treated with ribonuclease A prior to gel electrophoresis, it results in many different-sized bands. This indicates that during the labeling, intermediates are produced in which single-stranded DNAs of various lengths are associated with a compensatory length of RNA such that the total length for each intermediate is identical. These results provide evidence for the previously proposed model in which msDNA is synthesized by reverse transcriptase using a folded RNA precursor as a primer as well as a template. Furthermore, we found that there is a precise coupling mechanism of reverse transcriptase and ribonuclease H.

Cell-Free System↗
PubMed1989

Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus.

Myxobacteria have been shown to produce a peculiar RNA-DNA complex called msDNA, in which a single-stranded DNA is branched out from a RNA molecule (msdRNA) by a 2',5' phosphodiester linkage. It has been predicted that reverse transcriptase is required for msDNA biosynthesis. We identified a gene for reverse transcriptase in M. xanthus in the region that has been demonstrated to code for a cis- or transacting element for msDNA synthesis. This gene is located immediately downstream of the msdRNA coding region, and codes for a polypeptide of 485 amino acid residues. The polypeptide shows sequence similarity with retroviral reverse transcriptases. This fact, together with the mode of msDNA synthesis, suggests a possible relationship between retroviruses and the msDNA system. The analysis of the gene and the distribution of the msDNA system in independent isolates of M. xanthus indicate that the element is as old as other essential genes in M. xanthus and that it was not recently acquired into the genome.

Amino Acid Sequence↗
PubMed1989

Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA.

Branched RNA-linked multicopy single-stranded DNA (msDNA) originally detected in myxobacteria has now been found in a clinical isolate of Escherichia coli. Although lacking homology in the primary structure, the E. coli msDNA is similar in secondary structure to the myxobacterial msDNA's, including the 2',5'-phosphodiester linkage between RNA and DNA. A chromosomal DNA fragment responsible for the production of msDNA was cloned in an E. coli K12 strain; its DNA sequence revealed an open reading frame (ORF) of 586 amino acid residues. The ORF shows sequence similarity with retroviral reverse transcriptases and ribonuclease H. Disruption of the ORF blocked msDNA production, indicating that this gene is essential for msDNA synthesis.

Amino Acid Sequence↗
PubMed1989

Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli.

EnvZ and OmpR, the regulatory proteins for ompF and ompC expression in Escherichia coli, belong to a modulator-effector family of regulatory proteins which are essential for the response to environmental signals. We show that the soluble cytoplasmic domain of the transmembrane modulator protein EnvZ is phosphorylated in vitro by [gamma-32P]-ATP. We also demonstrate that the phosphate group can, in turn, be transferred to the transcription activator protein OmpR. The pH stability properties of the phosphate groups linked to EnvZ indicate that this molecule contains histidyl phosphate. The invariant His-243 of EnvZ corresponds to the phosphorylated His-48 of the chemotactic modulator protein CheA. Substitution of His-243 with valine produces an EnvZ that is refractory to phosphorylation and can no longer catalyze the transfer of phosphate to OmpR. Furthermore, in a delta envZ strain of E. coli, containing the envZ Val-243 plasmid, ompC expression is elevated 7-fold relative to that found in cells carrying the wild-type envZ plasmid. Based on these results we propose a model in which the phosphorylated state of OmpR modulates the expression of the ompF and ompC genes.

Bacterial Outer Membrane Proteins↗
PubMed1989

Extensive diversity of branched-RNA-linked multicopy single-stranded DNAs in clinical strains of Escherichia coli.

Recently it was shown that a clinical strain of Escherichia coli contains a reverse transcriptase that is essential for the synthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA). We now have examined 113 independent clinical isolates of E. coli for the existence of msDNA and found that 7 strains contained msDNA. Four of them were further analyzed by hybridization analysis, which indicated that three of the msDNAs were different, having little sequence homology. When the reverse transcriptase gene associated with one of these msDNAs was used as a probe, it did not hybridize with chromosomal DNA from the other strains containing msDNA. These results indicate that some clinical E. coli strains carry their own unique msDNA-synthesizing systems; msDNAs produced by these systems have little, if any, sequence homology in their RNA and DNA molecules and the reverse transcriptases required for the production of msDNA also have little sequence similarity. Such extensive diversity of the msDNA-synthesizing systems supports the notion that they were acquired by the E. coli genome late during the evolution of E. coli.

Blotting, Southern↗
PubMed1989

The role of Pro-239 in the catalysis and heat stability of subtilisin E.

Site-directed mutagenesis was employed to analyze the role of an alpha-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the alpha-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the alpha-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the alpha-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.

Catalysis↗
PubMed1989

Artificial immune system against viral infection involving antisense RNA targeted to the 5'-terminal noncoding region of coliphage SP RNA.

We previously reported the utilization of antisense RNA in the development of a novel immune system against RNA coliphage SP proliferation (Hirashima et al. [1986] Proc. Natl. Acad. Sci. U.S. 83, 7726-7730). We attempted to determine the most effective (i.e., those eliciting antiviral activity) sequences for targeting micRNAs within the 5'-terminal noncoding region of 54 nucleotides (nt). It was found that a 30-nt micRNA against the sequence from base 32 to 61 exhibited nearly complete inhibition of phage production. Upon further dissection of this sequence, it was concluded that the most effective micRNA against phage SP production should contain the sequences complementary to the Shine-Dalgarno (SD) sequence of the first gene and its 13-nt upstream sequence. The addition of downstream sequences had little effect. These results suggest that the micRNA functions by preventing the binding of ribosomes to the SD sequence of the first gene. The addition of further upstream sequences had a significant negative effect on the micRNA function, indicating that the removal of such impeditive sequences from a micRNA is an important strategy for the development of a potent micRNA immune system.

Base Sequence↗
PubMed1989

DNA-binding properties of the transcription activator (OmpR) for the upstream sequences of ompF in Escherichia coli are altered by envZ mutations and medium osmolarity.

Expression in Escherichia coli of the genes that encode the major outer membrane porin proteins (OmpF and OmpC) is regulated by the transcription activator protein OmpR and the receptorlike protein EnvZ, which is located in the inner membrane. Using synthesized oligonucleotide fragments containing the OmpR-binding site of ompF, we show that soluble extracts and partially purified OmpR derived from both the parent strain grown in nutrient broth plus 20% sucrose and the envZ11 strain grown in nutrient broth produced high-affinity DNA-binding activity, whereas soluble extracts from the parent strain grown in nutrient broth produced low-affinity binding. We also show that the soluble extracts from the envZ22(Am) strain grown in nutrient broth did not produce detectable bound forms of the ompF fragments, but low levels of DNA binding were detected with soluble extracts of the envZ22 strain grown in nutrient broth plus sucrose. In addition, the time course of the repression of OmpF synthesis produced by a shift to high-osmolarity growth medium was correlated with an increase in the DNA-binding affinity of soluble extracts to the ompF fragment. These results provide evidence that envZ function influences the DNA-binding activity of OmpR and suggest that high-affinity binding of OmpR to the upstream sequences of ompF is correlated with the repression of OmpF production.

Bacterial Outer Membrane Proteins↗
PubMed1988

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis.

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.

Bacillus subtilis↗
PubMed1988

Antisense RNA: its functions and applications in gene regulation--a review.

All known antisense RNAs existing in nature are described. Of 11 natural antisense RNAs, nine function at the level of transcription and two at the level of DNA replication. On the basis of their inhibitory mechanisms they can be separated into three classes. From what can be found in the naturally occurring antisense RNAs, strategies in designing artificial antisense RNAs for gene expression are proposed, and applications and potential problems discussed.

DNA Replication↗
PubMed1988

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein.

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.

Amino Acid Sequence↗
PubMed1988

In vitro processing of pro-subtilisin produced in Escherichia coli.

In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.

Bacterial Proteins↗
PubMed1988

A family of small repeated elements with some transposon-like properties in the genome of Neisseria gonorrhoeae.

A physical technique known as two-dimensional S1 nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae. This has resulted in the detection of two novel types of repetitive sequences. The first type is a repetitive sequence family of 152 base pairs (bp), whose ends are composed of inverted repeats of 26 bp. There are approximately 20 copies of this sequence, in both N. gonorrhoeae and Neisseria meningitidis (Correia, F., Inouye, S., and Inouye, M. (1986) J. Bacteriol. 167, 1009-1011). The second type of sequence is a 1443-bp duplication in the N. gonorrhoeae genome. The two classes of sequence are linked positionally. Each copy of the long duplicated sequence is adjacent to a member of the 152-bp repetitive sequence. In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable element.

Base Sequence↗
PubMed1988

Signal peptidases recognize a structural feature at the cleavage site of secretory proteins.

The cloning of the gene for staphylococcal nuclease A in the pIN-III-OmpA secretion vector results in a hybrid protein which is processed by signal peptidase I, yielding an active form of the nuclease that is secreted across the cytoplasmic membrane (Takahara, M., Hibler, D., Barr, P. J., Gerlt, J. A., and Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674). Using oligonucleotide-directed site-specific mutagenesis, we have constructed a set of mutants at the cleavage site area of the precursor hybrid protein designed to alter progressively the predicted secondary structure of the cleavage site. Our results show that processing becomes increasingly defective as the turn probability decreases. These results are consistent with the structural requirement that we found for the processing of lipoprotein by signal peptidase II (Inouye, S., Duffaud, G., and Inouye, M. (1986) J. Biol. Chem. 261, 10970-10975). We conclude that secretory precursor proteins have a distinct secondary structural requirement at their cleavage site for processing by signal peptidase I, as well as by signal peptidase II.

Amino Acid Sequence↗
PubMed1988

Modulation of the effects of mutations in the basic region of the OmpA signal peptide by the mature portion of the protein.

Oligonucleotide-directed site-specific mutagenesis was used to study the structure-function relationship of the positively charged amino terminus of the Escherichia coli outer membrane protein OmpA signal peptide. Mutations were isolated which reduced the overall charge of the amino-terminal region from +2 (wild type) to +1, 0, and -1, as well as one mutation from Thr to Ser at position 4. DNA encoding the wild type and mutant OmpA signal peptides was then fused in-frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. In the case of both the beta-lactamase and nuclease fusions, normal processing was no longer observed when the charge at the amino terminus was reduced to zero or made negative. Differences between the two hybrid proteins were observed in the case of the Thr to Ser mutation. As expected, this mutation had no effect on the beta-lactamase hybrid; however, the processing rate of the nuclease hybrid protein was reduced to nearly one-half. Furthermore, this effect was essentially reversed when a Lys residue at position 3 was deleted. A model is presented which explains the differing effects of a signal peptide mutation on the secretion of different hybrid proteins based on kinetic differences in the translocation of the nuclease and beta-lactamase proteins.

Amino Acid Sequence↗
PubMed1988

A new species of multicopy single-stranded DNA from Myxococcus xanthus with conserved structural features.

Myxobacteria have been shown to contain a large number of branched RNA-linked single-stranded DNA (multicopy single-stranded DNA (msDNA] molecules. In addition, we found that Myxococcus xanthus contains another smaller msDNA-like molecule, designated mrDNA, consisting of a 65-base single-stranded DNA covalently linked by a 2',5'-phosphodiester linkage to a 49-base branched RNA. In spite of their different primary sequences, the RNA-linked mrDNA is remarkably similar in secondary structure to msDNA, sharing similar stem-loop folding as well as the unique 2',5'-phosphodiester linkage. These results indicate that these novel molecules are synthesized by common molecular mechanisms.

Base Sequence↗
PubMed1988

A single amino acid determinant of the membrane localization of lipoproteins in E. coli.

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.

Amino Acid Sequence↗