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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 451 records · Page 25Linked to original sources
PubMed1982

Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli.

The gene for the outer membrane lipoprotein (lpp), the most abundant protein of Escherichia coli, was used to construct multi-purpose expression cloning vehicles. These vehicles consist of two types in terms of gene expression, one for constitutive (pIN-I type; three vehicles) and the other for inducible (pIN-II type; three vehicles) gene expression, and have the following features: (a) The lpp gene was inserted into a multicopy plasmid, pBR322, and the tet gene was removed to keep the size of the vehicles minimal (approximately 5 kb). (b) A nucleotide sequence of 22 bp which contains EcoRI, HindIII, and BamHI sites was inserted at the position of the third amino acid of the prolipoprotein. (c) The same nucleotide sequence was also inserted in two other reading frames at the same position. There are no other EcoRI, HindIII, and BamHI sites in the vehicles. Therefore, six different types of restriction fragments (EcoRI-EcoRI, HindIII-HindIII, BamHI-BamHI, EcoRI-HindIII, EcoRI-BamHI, and HindIII-BamHI) can be cloned at this position in any of the three different reading frames. (d) The nucleotide sequence from position 46 to 168 of the lpp gene was deleted. However, the 3' end position of the lpp gene of 154 bp was retained, which contains not only translation termination codons in three different reading frames but also the transcription termination signal of the lpp gene. Thus, this sequence is assumed to prevent unnecessary translation as well as transcriptional read-through of a cloned gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Effects of 2.45 GHz microwave radiation on the development of Japanese quail cerebellum.

Fertilized Japanese quail eggs were continuously exposed to 2.45 GHz microwave radiation from day 1 through day 12 of incubation at a power density of 5.0 +/- 0.52 mW/cm2 (mean specific absorption rate = 4.03 mW/gm). In the experiment with embryos, irradiated and control embryos were removed from eggs on day 12, 13 or 14 of incubation and the cerebella were histologically examined. In order to examine the long-term effect of microwave radiation during embryogenesis on the cerebellum, some of the quail were allowed to hatch and were reared to eight weeks of age. Their cerebella were histologically examined, and the extent of dendritic arbores, the length of the stem of the primary dendrite, and the size of the perikaryon of Purkinje cells were measured in Golgi-Cox impregnated sections. In the irradiated embryos, a slight developmental retardation was found in the cerebellar cortices in terms of several morphological parameters. The effects included the growth and subsequent decline of the external granular layer, the growth of the molecular layer, the cellular differentiation and the alignment of Purkinje cells, and the accumulation of granule cells beneath the Purkinje cell layer, as well as lower body and brain weights. In the eight-week-old quail, no significant differences were noted between irradiated and control cerebella in the morphological measurements of Purkinje cells.

Animals↗
PubMed1982

Effects of environmentally differential rearing upon maze performance in prenatally irradiated microcephalic rats.

Pregnant rats received 100 rads of X-irradiation on day 17 of gestation. Control pregnant rats were sham-irradiated on the same gestation day. The male offspring were reared under environmentally enriched, standard colony, and impoverished conditions for 30 days after weaning. Then the Hebb-Williams maze test was carried out. All the prenatally X-irradiated rats were microcephalic: their mean cerebral wet weight was 15.5% less than controls. The effect of X-irradiation was not significant in error scores and running times, whereas the effect of environment was significant in these items; initial and total error scores and running times were decreased in enriched groups compared to impoverished groups in controls as well as in X-irradiated animals.

Animals↗
PubMed1982

Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane.

The positively charged amino-terminal region of the signal peptide has been proposed to have an important role at an initial step of protein secretion across the membrane (loop model). To test this hypothesis, the charge on the amino-terminal region of the signal peptide of the prolipoprotein of the Escherichia coli outer membrane was altered by using synthetic oligonucleotides from +2 to +1, 0, and -1 by guided site specific mutagenesis of a plasmid DNA carrying an inducible lipoprotein gene. The wild-type sequence of this sectio, Met-Lys-Ala-Thr-Lys (+2), was thus changed to Met-Lys-Asp-Thr-Lys (I-1; +1), Met-Ala-Thr-Lys (I-2; +1), Met-Asp-Thr-Lys (I-3; 0), and Met-Glu-Asp-Thr-Lys (I-4; -1). After induction of lipoprotein production, cells were pulse labeled with [35S]methionine for 10 sec. The lipoprotein of I-1, I-2, and I-3 was assembled in the membrane, although the rates of lipoprotein production progressively decreased as the charge on the signal peptide became more negative. Conversely, in the case of I-4, only a small amount of lipoprotein assembled in the membrane while a large amount of glycerol-unmodified prolipoprotein accumulated in the cytoplasm. This soluble prolipoprotein was gradually and posttranslationally secreted across the membrane to be modified and assembled in the membrane. These results indicate that the positively charged amino-terminal region of the signal peptide plays an important role in efficient protein secretion across the membrane.

Amino Acid Sequence↗
PubMed1982

Unusually long mRNA species coding for human alpha and beta interferons.

Each of the several intronless interferon (IFN) alpha S genes is embedded in 4- to 5-kilobase (kb) DNA homology units which are arranged more or less in tandem in the human genome. In addition to the expected 0.8- to 1.4-kb-long IFN-alpha S mRNA species, we have detected polyadenylylated RNA molecules approximately 2-2.7, 3.5-4.5, and 7-8 kb long derived from IFN-alpha S genes in the cytoplasm of Sendai virus-induced bromodeoxyuridine-treated human lymphoblastoid (Namalwa) cells. These transcripts were detected by electrophoresis of cytoplasmic polyadenylylated RNA through agarose-CH3HgOH gels followed by blothybridization using an IFN-alpha 1 (a prototype alpha S) cDNA probe and also by translation of the eluted RNA into biologically active IFN in the Xenopus oocyte assay. At least some of these long translationally active RNA molecules appear to represent functional mRNA species because they can be detected in polysomes. In addition, the oocyte translation assay has revealed the presence of unusually long (2-8 kb) IFN-beta mRNA species both in Sendai virus-induced Namalwa cells and in poly(I) . poly(C)-induced diploid human fibroblasts (FS-4). The detection of very long IFN-alpha 1-related mRNA species suggests that some of the DNA sequence features observed in the flanking homology units may affect the transcription of the IFN-alpha S genes. Our hybridization analyses also indicate that the 1.8-kb human IFN-alpha L mRNA(s) shows no detectable cross-hybridization with an IFN-alpha 1 cDNA probe. Thus, this mRNA is derived from a gene(s) that is distinct from the IFN-alpha S set.

Cell Line↗
PubMed1982

Cloning of the regulatory genes (ompR and envZ) for the matrix proteins of the Escherichia coli outer membrane.

We have cloned the regulatory gene cluster of Escherichia coli which is composed of at least two distinct genes, ompR and envZ. These genes are known to regulate the production of the outer membrane matrix proteins. The newly formed plasmids were found to complement not only ompR mutations but also envZ mutations. The ompR gene product was identified as a protein of an apparent molecular weight of 28,500.

Bacterial Outer Membrane Proteins↗
PubMed1982

Phosphorylation and methylation of proteins during Myxococcus xanthus spore formation.

Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.

Adenosine Triphosphate↗
PubMed1982

Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant.

A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated. The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive. The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.

Bacterial Outer Membrane Proteins↗
PubMed1982

Use of a lac promoter-operator fragment as a transcriptional control switch for expression of the constitutive lpp gene in Escherichia coli.

We constructed hybrid plasmids to allow controlled expression of the lpp gene coding for the outer membrane lipoprotein of Escherichia coli, which is otherwise expressed constitutively. This was achieved by the insertion of a DNA fragment carrying the lacUV5 promoter-operator region as a transcriptional control switch into the 5'-untranslated region of the lpp gene. When fully induced, the production of the lipoprotein, controlled under the tandem promoters of lppp-lacpo-lpp, increased approximately 3-fold compared to that under lacpo-lpp control. However, it was still only one-third of the lipoprotein production under the constitutive lpp expression. One such plasmid, pKEN125, carrying lppp-lacpo-lpp in pBR322 produced only a trace amount of the lipoprotein without induction in an E. coli lpp- cell. Upon the addition of isopropyl-beta-d-thiogalactoside, however, the amount of the lipoprotein reached almost 40% of the total membrane proteins. Cells carrying pKEN125 grew normally in the presence of the inducer, whereas cells carrying plasmid pKEN126 with tandem duplication of lppp-lacpo-lpp sequences in pBR322 lysed upon induction at high temperature. In cells with pKEN126 induced at high temperature, at least three new bands which were cross-reactive with antilipoprotein serum in addition to the mature lipoprotein were detected by pulse-labeling cells with [35S]methionine.

Base Sequence↗
PubMed1981

Accumulation of the prolipoprotein of the Escherichia coli outer membrane caused by benzyloxycarbonylalanine chloromethyl ketone.

Benzyloxycarbonylalanine chloromethyl ketone (Z-Ala-CH2Cl) was found to inhibit the processing of the prolipoprotein, a secretory precursor of the lipoprotein of the Escherichia coli outer membrane. The accumulation of the prolipoprotein in the presence of Z-Ala-CH2Cl was observed both in vivo and in vitro. Z-Ala-CH2Cl did not inhibit the processing of precursors of the other major outer membrane proteins. A preliminary experiment to identify the processing enzyme(s) of the prolipoprotein was carried out with use of Z-[3H]Ala-CH2Cl and revealed that two cytoplasmic membrane proteins with Mr = 78,000 and 46,000 were predominantly labeled.

Amino Acid Chloromethyl Ketones↗
PubMed1981

Comparison of the lipoprotein gene among the Enterobacteriaceae. DNA sequence of Erwinia amylovora lipoprotein gene.

A DNA sequence of 816 base pairs encompassing the entire Erwinia amylovora lipoprotein gene was determined. Sequence comparison between E. amylovora, Escherichia coli, and Serratia marcescens suggests that the structure of the lipoprotein has been highly conserved under the constraint of efficient gene expression selecting promoter structure, mRNA secondary structure, and codon usage in addition to the polypeptide function. The sequence also suggests that the lpp gene of the three bacteria diverged sequentially in the course of evolution.

Base Sequence↗
PubMed1981

Avoidance learning under delayed shock termination in prenatally x-irradiated rats.

Groups of MPI male albino rats were exposed to x-irradiation of 200 rad on Day 17 of gestation. Irradiated and control rats were trained in a shuttle box with either immediate or .5-sec delayed termination of both the electric shock (US) and the conditioned stimulus (CS) on escape trials. An avoidance response always produced prompt CS termination. Irradiated rats showed a higher degree of avoidance behaviour than controls and, under the delay condition, tended to run rapidly in the CS-US interval.

Animals↗
PubMed1981

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points.

A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp- cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp- mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From resistance enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occurred in the A-T rich 5' untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.

Bacterial Proteins↗
PubMed1981

Biosynthesis of membrane proteins of Pseudomonas aeruginosa: effects of various antibiotics.

The biosynthesis of membrane proteins of Pseudomonas aeruginosa was examined using various antibiotics (puromycin, streptomycin, chloramphenicol, tetracycline, and rifampin). Among six major membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the biosynthesis of two membrane proteins (proteins I and II) was found to be unusually resistant to these antibiotics. The biosynthesis of protein I (apparent molecular weight of 6,500) was completely resistant to puromycin, streptomycin, chloramphenicol, and tetracycline at conditions which severely inhibited the biosynthesis of all the other membrane proteins except for protein II. Under the same conditions, the biosynthesis of protein II (apparent molecular weight of 9,000) was also resistant to puromycin, streptomycin, and tetracycline, but was sensitive to chloramphenicol. The effect of rifampin on the biosynthesis of proteins I and II indicated that their messenger RNAs are extremely stable; their functional half-lives are 16 and 8 min for proteins I and II, respectively, in contrast with 2.0 and 3.5 min for the average half-lives of the cytoplasmic and membrane proteins, respectively. Protein II was identified as the lipoprotein of the outer membrane from its amino acid composition and mobility in gel electrophoresis. Protein I is a cytoplasmic membrane protein lacking histidine. From the content of arginine residues, the number of protein I molecules per cell was estimated to be as many as, and most likely more than, that of the lipoprotein (protein II). Therefore, protein I is the most abundant protein in P. aeruginosa.

Anti-Bacterial Agents↗
PubMed1981

Preferential selection of deletion mutations of the outer membrane lipoprotein gene of Escherichia coli by globomycin.

Globomycin is an antibiotic which inhibits the processing of the prolipoprotein. Eighty globomycin-resistant mutants were independently isolated from Escherichia coli K-12 which had a deletion mutation in chromosomal lipoprotein gene (lpp), but contained a plasmid carrying the wild-type lpp gene. Twenty-six of the mutants did not have the lipoprotein in the membrane fractions. From the analysis of the plasmids of these mutants, all of the lipoprotein-deficient mutations were found to be due to deletion mutations around the lpp gene.

Anti-Bacterial Agents↗
PubMed1981

Reexamination of the genome size of myxobacteria, including the use of a new method for genome size analysis.

The genome sizes of two myxobacteria, Myxococcus xanthus and Stigmatella aurantiaca, were measured by renaturation analysis and also by a new method involving the quantitation of individual restriction fragments. In contrast to several previous reports, which indicate that M. xanthus has a genome size which is three to four times that of Escherichia coli, the present measurements indicated that the M. xanthus genome is only about 24 to 53% larger than that of E. coli. S. aurantiaca had a genome size nearly identical to that of M. xanthus. Of possible significance is the fact that the renaturation curves of M. xanthus and S. aurantiaca deoxyribonucleic acid both exhibited significant fractions which renatured with rapid, unimolecular kinetics. However, we were unable to establish that these fractions represented inverted repeats of repetitive sequences.

Chromosomes, Bacterial↗
PubMed1981

Expression of the Serratia marcescens lipoproteins gene in Escherichia coli.

The lipoprotein gene (lpp) of Serratia marcescens was cloned in a lambda phage vector (K. Nakamura and M. Inouye, Proc. Natl. Acad. Sci. U.S.A. 77: 1369-1373, 1980). This lpp gene was recloned in plasmid vectors pBR322 and pSC101. When a lipoprotein-deficient (lpp) mutant of Escherichia coli was transformed with pBR322 carrying the S. marcescens lpp gene, cells became nonleaky for ribonuclease, resistant to ethylenediaminetetraacetic acid, and sensitive to globomycin. The lipoprotein was found exclusively in the outer membrane fraction. These results indicate that the S. marcescens lipoprotein was normally secreted across the cytoplasmic membrane, modified, and assembled in the E. coli outer membrane. The amount of the free-form lipoprotein produced in this system was three times higher than that produced in lpp + C. coli cells, whereas there was no difference in the amount of the bound-form lipoprotein. On the other hand, lpp E. coli cells which harbored pSC101 carrying the S. marcescens lpp gene produced only one-third of the free-form lipoprotein produced in lpp E. coli cells which harbored pSC101 carrying the E. coli lpp gene. One of the major factors causing this difference in efficiency of gene expression between the lpp genes of S. marcescens and E. coli appears to be a deletion mutation at the transcription termination region found in the cloned S. marcescens lpp gene. The functional half-life of the S. marcescens lpp messenger ribonucleic acid in E. coli was found to be found half that of the E. coli lpp messenger ribonucleic acid.

Cell Membrane↗