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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 235 records · Page 13Linked to original sources
PubMed1993

Oar, a 115-kilodalton membrane protein required for development of Myxococcus xanthus.

Myxococcus xanthus is a developmental gram-negative bacterium which forms multicellular fruiting bodies upon nutrient starvation. This bacterium was found to contain a 115-kDa membrane protein which separated with the inner membrane fraction by sucrose density gradient centrifugation. The gene for this protein was cloned, and its DNA sequence was determined. The deduced amino acid sequence consists of 1,061 residues. This protein contains a putative signal sequence and many short segments, found scattered throughout the entire protein, that have sequence similarities with OmpA, a major outer membrane protein of Escherichia coli. Thus, the gene was designated oar (OmpA-related protein). A second open reading frame was found 36 bases downstream of the oar termination codon. This open reading frame encodes a protein of 236 residues and contains a putative lipoprotein signal sequence. An aor disruption mutation (delta oar) showed no effect on vegetative growth but caused abnormal morphogenesis during development and reduced myxospore formation. When examined with a light microscope, delta oar cells were unable to aggregate on developmental agar, indicating that Oar is required for cellular adhesiveness during development.

Amino Acid Sequence↗
PubMed1993

Chloramphenicol induces the transcription of the major cold shock gene of Escherichia coli, cspA.

A downshift in temperature or exposure of cells to certain inhibitors of translation has been shown to induce the synthesis of cold shock proteins in Escherichia coli. We characterized the induction of the major cold shock protein (CS7.4, the product of the cspA gene) of E. coli in response to a shift from 37 to 15 degrees C and in response to the addition of chloramphenicol at 15 degrees C. A pulse-labeling assay and primer extension experiments indicated that the cold shock treatment resulted in a transient increase in the level of the cspA transcript and a transient induction of CS7.4, while the addition of chloramphenicol resulted in a constitutive increase in the level of cspA transcript and constitutive production of CS7.4. The addition of rifamycin immediately following the temperature downshift or along with the addition of chloramphenicol repressed the transcription of cspA as well as the induced production of CS7.4. Furthermore, changes in the cspA mRNA level were coincident with changes in CS7.4 synthesis. These results indicate that the expression of cspA induced by cold shock and chloramphenicol is at the level of transcription but not at the level of translation. Measurement of the half-life revealed that the cspA mRNA induced by chloramphenicol was more stable than that induced by cold shock.

Bacterial Proteins↗
PubMed1993

Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus.

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.

Adenosine Triphosphate↗
PubMed1993

Phenotypic revertant mutations of a new OmpR2 mutant (V203Q) of Escherichia coli lie in the envZ gene, which encodes the OmpR kinase.

The Escherichia coli ompR2 allele ompR472 contains a valine-to-methionine point mutation at position 203, resulting in an OmpF-constitutive OmpC- outer membrane phenotype. In the present study, OmpR residue V-203 was replaced with glutamine (V203Q mutation), resulting in the same outer membrane phenotype. However, unlike the OmpFc OmpC- phenotype conferred by the OmpR(V203M) mutant protein, the OmpFc OmpC- phenotype produced by the OmpR(V203Q) mutation was suppressed by the envZ11(T247R) allele. Additional suppressors of OmpR(V203Q) were isolated by random mutagenesis. All suppressor mutations were found in the envZ gene and conferred an OmpC+ OmpF- phenotype in the presence of the wild-type ompR. These envZ11-like mutations mapped to a region different from those previously reported and were incapable of suppressing the ompR(V203M) allele. Our results indicate that while methionine or glutamine replacements could cause similar effects on OmpF and OmpC expression, they conferred different abilities on the mutant proteins to be suppressed by envZ.

Bacterial Outer Membrane Proteins↗
PubMed1993

Disturbance of neuronal migration in mouse cerebral cortex by low-dose gamma-radiation.

We show that neuronal migration is disturbed by low-dose gamma-radiation of 0.24 Gy in the developing cerebral cortex of mice and suggest that neuronal progenitors in the S phase of mitosis are more sensitive than those in the G1/G0 phase. To pulse-label the fetal cells pregnant Slc:ICR mice were injected with bromodeoxyuridine (BrdU) at 10.00 pm on day 16 of pregnancy or at 9.30 am on day 17. The mice then were exposed to 0.24 Gy gamma- or sham-irradiation at 10.00 am on day 17 of pregnancy. At the time of exposure cells labeled on day 16 were regarded as having completed mitosis, and those labeled on day 17 as being in the S phase. Cell death in the fetal ventricular zone after exposure was negligible. Dams were allowed to give birth and rear their litters. Brain samples obtained from offspring on the 2nd day after birth and at 6 weeks of age were used for the immunohistochemical examination of BrdU-labeled cells. Labeled cells remaining in the ventricular zone were significantly more numerous in irradiated animals than in sham-exposed ones on the 2nd day after birth, in mice treated prenatally with BrdU on day 17 of pregnancy; whereas, mice treated with BrdU on day 16 showed no statistically significant difference. At 6 weeks of age, in both groups most of the labeled cells were present in layers II-III of the SmI cerebral cortex. But, in the irradiated animals the labeled cells in these layers were significantly fewer and those in layers IV-VI more numerous than in the sham-exposed mice in both group treated with BrdU on day 16 or 17.

Animals↗
PubMed1993

One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.

A simple and efficient method was developed for insertion of oligonucleotide sequences into plasmids. In this method, an unphosphorylated oligonucleotide was ligated to the restriction-digested phagemid DNA. Only the single strand of the oligonucleotide was ligated at the 5' end of the phagemid, and this resulted in the creation of a long self-complementary single-strand overhang. These single-strand overhang-possessing phagemids were used to transform XL-1 cells. This simple ligation and transformation reaction rendered approximately 7.5 x 10(4) to 5 x 10(5) of white colonies per microgram DNA from the isopropyl-beta-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside plate. This number is almost the same or even higher than the number of blue colonies from the control reaction in which ligase was used without the oligonucleotide. By this method we could mutate one enzyme site to another or create ribozyme and substrate phagemid very easily. Fidelity of this method was checked by restriction digestion, DNA sequencing and ribozyme reaction. By DNA sequencing, we observed that 100% of the white colonies contained a single oligonucleotide sequence.

Base Sequence↗
PubMed1992

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.

Amino Acid Sequence↗
PubMed1992

Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.

Amino Acid Sequence↗
PubMed1992

Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase.

A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli. The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras. The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan). The strain was found to be cold-sensitive for growth. Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein.

Bacterial Proteins↗
PubMed1992

Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing.

We have identified a yeast gene encoding a protein structurally similar to mammalian nucleolin. The gene was previously cloned as a cold shock-inducible gene and found to be identical to yeast NSR1 gene, which encodes a protein that has been reported to bind sequences required for nuclear localization of protein. The carboxyl-terminal half of NSR1, consisting of two tandemly repeated putative RNA-binding domains and a glycine/arginine-rich domain, has 37% amino acid sequence identity with the same part of mammalian nucleolin, while no sequence similarities are found between their amino-terminal regions. Although a null mutation of the NSR1 gene was not lethal, it caused a severe defect on growth. Pulse-labeling analysis revealed that the nsr1 strain had reduced levels of 18 S rRNA and accumulated 35 S pre-rRNA compared with the wild-type strain. The level of 25 S rRNA was also slightly reduced in the nsr1 strain. Pulse-chase labeling experiments showed slow processing of 35 S pre-rRNA and impaired methylation of 18 S rRNA. The ratio of 40 S to 60 S ribosomal subunits in the nsr1 strain is significantly reduced and is consistent with impaired synthesis of 18 S rRNA. The results indicate that NSR1 is involved in pre-rRNA processing and ribosome biosynthesis in yeast.

Amino Acid Sequence↗
PubMed1992

Cold shock induction of yeast NSR1 protein and its role in pre-rRNA processing.

Yeast NSR1 protein is structurally related to mammalian nucleolin and is involved in ribosome synthesis as described in the preceding paper (Kondo, K., and Inouye, M. (1992) J. Biol. Chem. 267, 16252-16258). We report here the regulation of NSR1 gene expression and the effect of nsr1 deletion on growth and pre-rRNA processing after cold shock. A basal level of the transcript was detected at 30 degrees C only in exponentially growing cells and increased approximately 3-fold after cold shock to 10 degrees C. NSR1 protein level also increased about 3-fold after the shock. The nsr1 deletion caused a severe growth defect at low temperatures as well as a long growth lag after temperature shift from 30 to 10 degrees C. Northern hybridization analysis demonstrated that cold shock led to a rapid decrease in the amounts of 27 S, 20 S, and 7 S rRNA precursors in the nsr1 strain, although at 30 degrees C a major defect was observed only in 20 S pre-rRNA synthesis. Pulse-chase labeling of rRNA showed that pre-rRNA processing in the nsr1 strain was greatly impaired after cold shock. These results demonstrate that the NSR1 protein is required for normal pre-rRNA processing and cell growth in yeast when cells are exposed to abrupt temperature drop.

Base Sequence↗
PubMed1992

Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.

msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).

Base Sequence↗
PubMed1992

In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron.

Gram-negative bacteria such as Myxococcus xanthus, Stigmatella aurantiaca, and Escherichia coli contain retroelements called retrons. Retrons consist of the msr-msd region and the gene for reverse transcriptase (RT), which are essential for the production of the branched RNA-linked ms-DNA (multicopy single-stranded DNA). In this study, we attempted to produce msDNA in the yeast Saccharomyces cerevisiae. Retron Ec67 from E. coli, which is responsible for the production of msDNA-Ec67, was cloned under the GAL10 promoter in a 2-microns-based plasmid. msDNA thus produced was detected by extending the 3' end of the msDNA by avian myeloblastosis virus RT. This yielded a main product of 117 nucleotides. Treatment of this product with RNase A resulted in a DNA of 105 nucleotides. These results are in good agreement with the structure of msDNA-Ec67. The production of msDNA-Ec67 was further confirmed by Southern blot hybridization. The msDNA production was dependent upon the bacterial RT gene in the clone and was increased severalfold when the RT gene of retron Ec67 was placed in front of the msr-msd region. The potential of msDNA as a eukaryotic vector producing a stable single-stranded DNA as well as RNA is discussed.

Base Sequence↗
PubMed1992

In vivo duplication of genetic elements by the formation of stem-loop DNA without an RNA intermediate.

Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in the diversification of genomes during evolution. Here, we demonstrate that a genomic DNA sequence can be duplicated in vivo as a result of template switching. When an inverted repeat (IR) structure was inserted in a site downstream from a ColE1 plasmid origin of DNA replication, transformation of Escherichia coli cells with this plasmid resulted in the production of a new DNA fragment encompassing the region from the origin to the center of the IR structure. The structure of this DNA molecule is composed of a long stem-loop formed by a single-stranded DNA, in which the loop is formed by the IR structure. The DNA fragment is designated slDNA, for stem-loop DNA. The experiments in this study suggest that during DNA replication, template switching at the stem-loop structure formed by the IR structure gives rise to slDNA utilizing the nascent DNA strand or the parental strand as a template. The mechanistic implications of slDNA synthesis, and its possible roles in genome evolution, are discussed.

Base Sequence↗
PubMed1992

A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.

Amino Acid Sequence↗
PubMed1992

Developmental alteration of serotonin neurons in the raphe nucleus of rats with methylazoxymethanol-induced microcephaly.

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation induces severe microcephaly in the offspring. The present study first investigated a developmental alteration of serotonin (5HT) neurons immunohistochemically in the dorsal and median raphe nuclei in serial sections in both control and microcephalic rats (MAM-rats) at 35 days of age. 5HT-immunoreactive neurons in the MAM-rats were reduced in number and irregularly distributed in the dorsal and median raphe nuclei compared with those in the control. The dendrites of neurons in these nuclei in the MAM-rats were very short and twisted. A follow-up observation on the development of the cerebral cortex at 5, 9 and 28 days of age was performed using Nissl-stained preparations, which revealed a disorganized cell arrangement in the cerebral cortex of the MAM-rats at the very early postnatal period. Furthermore, the distribution of 5HT-immunoreactive fibers into the cerebral cortex was also examined using brains of 28 days of age. In MAM-rats of this age, abnormally tortuous 5HT-immunoreactive fibers were observed in the cerebral cortex. 5HT neurons in the raphe nuclei are known to project their ascending axons widely into the entire cortical area during the 1st postnatal week. Thus, the association of disorganized cortical cell arrangement and the hyperdense and tortuous distribution of 5HT-immunoreactive fibers in the cerebral cortex support the idea of target-dependent secondary degeneration of 5HT neurons in the dorsal and median raphe nuclei of the MAM-rats.

Animals↗