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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 487 records · Page 27Linked to original sources
PubMed1979

DNA sequence of the gene for the outer membrane lipoprotein of E. coli: an extremely AT-rich promoter.

The outer membrane lipoprotein is the most abundant protein in an E. coli cell. Its structural gene (Ipp) was cloned into a lambda phage vector and the nucleotide sequence of a DNA fragment of 814 bp encompassing the Ipp gene was determined. The promoter region of the gene was found to have the following features. First, a segment of 261 bp preceding the transcription initiation site (-1 to -261) has a very high AT content of 70%, in contrast to 53% for the mRNA region of 322 bp, 44% for a segment of 127 bp after the transcription termination site and 49% for the average AT content of the E. coli chromosome. Second, in particular, of the first 45 bp upstream from the transcription initiation site (-1 to -45), 36 bases (80%) are A or T. Third, there is a heptanucleotide sequence homologous to the "Pribnow box," eight bases apart from the transcription initiation site. Fourth, a sequence homologous to the "RNA polymerase recognition site" exists on both strands between positions -27 and -39. Finally, there is a long dyad symmetry centered at the transcription initiation site.

Adenine↗
PubMed1979

Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that has a complex life cycle including a temporal sequence of cellular aggregation, mound formation, and myxosporulation. During development, protein S (molecuar weight 23,000) is induced and accumulates in very large amounts. Protein S was found in the soluble fraction of early developmental extracts and in the insoluble fraction in later extracts. This insoluble form of protein S can be solubilized by the addition of 1 M NaCl at 0 degrees C to extracts from aggregated cells (mound stage) or by the addition of 1 M NaCl at 30 degrees C to mature spores. Salt extraction (1 M NaCl) of protein S from mature spores was partially inhibited by the addition of Mg(2+) and almost completely inhibited by the addition of Ca(2+). The viability of spores was not changed by a salt extraction that removed their protein S. Examination of thin sections of mature spores and extracted spores by electron microscopy suggested that the protein S-deficient spores lacked a spore surface coat about 300 A thick. Purified protein S will spontaneously self-assemble onto protein S-deficient spores after removal of the NaCl by dialysis or by addition of 10 mM Ca(2+) to undialyzed samples. Glycerol-induced spores did not contain protein S and did not serve as primers for assembly of protein S. Quantitation of the self-assembly process showed almost stoichiometric binding of protein S to the protein S-deficient spores until saturation at 3.3 x 10(6) molecules per spore, a value 1.35 times higher than the normal level of proteins S found in mature spores. Protein S in the "reconstituted" spores was as protease resistant and sonication resistant as the protein S of native spores. Electron microscopy of the reconstituted spores revealed the assembly of new material on the spore surface. Adjacent spores were sometimes observed to be fused to each other through a common protein S layer. These results suggest that protein S serves a function in spore-spore interaction in the fruiting body.

Bacteria↗
PubMed1979

Homology of the gene coding for outer membrane lipoprotein within various Gram-negative bacteria.

The mRNA for a major outer membrane lipoprotein from Escherichia coli was found to hybridize specifically with one of the EcoRI and one of the HindIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens, and Erwinia amylovora. However, among the Enterobacteriaceae, DNA from two species of Proteus (P. mirabilis and P. morganii) did not contain any restriction endonuclease fragments that hybridized with the E. coli lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter sp. HO1-N, Caulobacter crescentus, and Myxococcus xanthus. Envelope fractions from all bacteria in the family Enterobacteriaceae tested above cross-reacted with antiserum against the purified E. coli free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of Proteus, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family Enterobacteriaceae did not cross-react with anti-E. coli lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the Enterobacteriaceae.

Antigens, Bacterial↗
PubMed1979

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.

Bacterial Proteins↗
PubMed1978

Teratogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine in mice.

The teratogenic effect on the mouse fetus of a potently mutagenic and carcinogenic agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied. Pregnant mice were injected on one of gestation days 7-12 with an intraperitoneal dose of 40, 60 or 80 mg/kg of MNNG, and fetuses were examined on day 18 of gestation. Various malformations affecting the brain, face, vertebra, rib and limb appeared in high frequency. Brain malformations were the most predominant; hydrocephalus, hydromicrocephaly and microcephaly appeared in sequence with developmental stages of treatment, although exencephaly occurred infrequently. Cleft palate, sometimes associated with microglossia and micrognathia, and vertebral anomalies including tail defect were observed following almost every gestation-day treatment. Long-bone defects of fore- and hindlimbs were also predominant. Polydactyly, ectrodactyly and microdactyly frequently occurred together in the fore- or hindlimbs or both in sequence with treatment stages. Ectrodactyly and microdactyly appeared with greater frequency on the left than on the right in both fore- and hindlimbs, but polydactyly and long-bone defects appeared bilaterally.

Abnormalities, Drug-Induced↗
PubMed1978

Spin labeling of a cysteine residue of the Escherichia coli outer membrane lipoprotein in its membrane environment.

A method was developed to attach a spin label to a specific site on the structural lipoprotein of the Escherichia coli outer membrane in situ. This method takes advantage of the fact that the outer membrane of wild-type E. coli contains few residues reactive towards sulfhydryl reagents. A mutant E. coli strain has been isolated [Suzuki, H., Nishimura, Y., Iketani, H., Campisi, J., Hirashima, A., Inouye, M. & Hirota, Y. (1976) J. Bacteriol. 127, 1494-1501] in which the second position from the carboxy terminus of the lipoprotein is changed from arginine into a cysteine residue. The membrane fraction of this mutant was treated with N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)maleimide in the presence of EDTA and 2-mercaptoethanol. Spin label was found to be preferentially incorporated into the lipoprotein. The spectrum of the spin-labeled membrane shows two components, both arising from spin label at the same site near the carboxy terminus. The strongly immobilized component has a maximum hyperfine splitting value of 53 G, and the weakly immobilized component, 37 G. A fraction of the lipoprotein is covalently bound to the peptidoglycan layer through its carboxy-terminal lysine; the spectrum of the isolated bound form of the lipoprotein was identical to that of the free form. When the matrix protein, the other major outer membrane protein, was removed by mutation, the spectrum of the lipoprotein was altered, suggesting that these two proteins are closely associated.

Bacterial Proteins↗
PubMed1978

Homologous nucleotide sequences between prokaryotic and eukaryotic mRNAs: the 5'-end sequence of the mRNA of the lipoprotein of the Escherichia coli outer membrane.

The sequence of the first 89 nucleotides at the 5' end of the mRNA for the lipoprotein of the Escherichia coli outer membrane is: GCUACAUGGAGAUUAACUCAAUCU-AGAGGGUAUUAAUAAUGAAAGCUACUAAACUGGUACU-GGGCGCGGUAAUCCUGGGUUCUACUCUG. The sequence of the first 72 nucleotides was established by direct sequencing methods and was extended to 89 residues on the basis of the known sequences of oligonucleotides obtained from complete digestion of the mRNA by ribonuclease T1 or A and the known amino acid sequence of the prolipoprotein. The mRNA has an untranslated region of 38 residues before the initiation codon, AUG. A unique feature of the 5'-end sequence of the mRNA is that the sequence of 12 nucleotides (GUAUUAAUAAUG) prior to, and including, the initiation codon is the same as that found at the ribosome-binding site for 80S ribosomes in brome mosaic virus RNA4, a eukaryotic mRNA [Dasgupta, R., Shih, D., Saris, C. & Kaesberg, P. (1975) Nature 256, 624-628].

Bacterial Proteins↗
PubMed1978

Interaction between two major outer membrane proteins of Escherichia coli: the matrix protein and the lipoprotein.

The affinity to the matrix protein, one of the major outer membrane proteins of Escherichia coli, for the peptidoglycan was examined of extracting the cell envelope complex at 55 degrees C and 2% sodium dodecyl sulfate containing different amounts of NaCl. It was found that the matrix protein was extracted from the peptidoglycan of a mutant strain (lpo) that lacks another major membrane protein, the lipoprotein, at a lower NaCl concentration than was the matrix protein of the wild-type cell (lpo+). When the envelope fraction of the wild-type strain was treated with trypsin, which is known to cleave the bound-form lipoprotein from the peptidoglycan, the affinity of the matrix protein for the peptidoglycan decreased to the same level as that of the affinity of the matrix protein for the peptidoglycan of the mutant strain. It was further shown that the free-form lipoprotein was also retained in the matrix protein-peptidoglycan complex, although the extent of retention of the free form of the lipoprotein was less than that of the matrix protein. These results indicate that both the free and the bound forms of the lipoprotein are closely associated with the matrix protein and that the bound form of the lipoprotein plays and important role in the association between the matrix protein and the peptidoglycan.

Bacterial Proteins↗
PubMed1978

Gene dosage effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.

The gene dosage effects of the structural gene (lpp) for the lipoprotein of the Escherichia coli outer membrane were examined. A novel F-prime factor containing the lpp gene was constructed. The amount of the free-form lipoprotein in the merodiploid strain carrying the F-prime factor was found to be about two times as great as that in the corresponding haploid strain. On the other hand, the amount of the bound-form lipoprotein, which is vovalently linked to the peptidoglycan, was not significantly different in the merodiploid strain as compared with the corresponding haploid strain. The present results suggest that the lpp gene is expressed constitutively in contrast to another major protein of the E. coli outer membrane, tolG protein (protein II, D. B. Datta et al., J. Bacteriol. 128:834-841, 1976). The F-prime factor isolated may include a portion of the E. coli chromosome (located between 33 and 36 min on the genetic map) that is not covered by any other F-prime factor.

Bacterial Proteins↗
PubMed1978

Lipoprotein from Proteus mirabilis.

The biosynthesis of a Proteus mirabilis outer membrane protein of molecular weight of approximately 7,000 was found to be relatively resistant to puromycin and rifampin, as is the case for the Escherichia coli liporotein. Furthermore, the existence of the lipoprotein in P. mirabilis was indicated by a comparison of the amino acid compositions of the purified free and bound forms of this protein with those of the E. coli free and bound lipoproteins.

Amino Acids↗