Search PubMed⌕ Search

Biomedical subjects

I N Maruyama

Publications and source records attributed to I N Maruyama.

At least 19 recordsLinked to original sources
PubMed1997

Efficient epitope mapping by bacteriophage lambda surface display.

A bacteriophage lambda surface expression system, lambda foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase 1 to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries.

Amino Acid Sequence↗
PubMed1996

Surface display of proteins on bacteriophage lambda heads.

We have developed plasmid and phage vectors for the display of foreign proteins on the surface of bacteriophage lambda capsid by modifying the D gene which encodes the major head protein gpD. The vectors have multiple cloning sites, and permit colour selection and conditional chain termination for recombinants. Displayed proteins can be fused to either the N or C terminus of gpD by a peptide linker. The conditional chain termination scheme, via a host Escherichia coli suppressor activity, allows the fusion and assembly of homomultimeric proteins as well as control of the number of fusion proteins per phage particle. We have successfully displayed beta-lactamase, IgG-binding domains of the Staphylococcus aureus protein A, and beta-galactosidase by cloning the genes into the vector. The constructs express functionally active proteins fused to gpD that assemble into phage particles. These results suggest that gpD may be fused to many other peptides and proteins at their N or C terminus and the fusion products may be accessible on the surface of bacteriophage lambda particles.

Amino Acid Sequence↗
PubMed1995

A model for transmembrane signalling by the aspartate receptor based on random-cassette mutagenesis and site-directed disulfide cross-linking.

Extracellular information is transduced by transmembrane receptors into the inside of the cell across a membrane barrier. To understand the molecular basis of transmembrane signalling, we replaced the transmembrane segment 2 (TM2) of the Escherichia coli aspartate receptor, Tar, with random sequences that are 21 amino acid residues in length and consist of Arg, Gly, Ser, Cys, Val, Leu, Ile and Phe at each position. From this ensemble for recombinant molecules, functional receptors were recovered as clones that could bind aspartate and transmit a signal to the intracellular domain. Restricted average hydrophobicity values were observed for functional transmembrane domains, and support the observation that transmembrane segments typically have hydrophobicity values greater than 1.6. However, non-functional transmembrane domains with greater hydrophobicity than 1.6 indicate that hydrophobicity is not a sole determinant for its function. Fourier transform analysis of the functional TM2 sequences suggests that the transmembrane segment has an alpha-helical structure with three distinct faces. Cross-linking of the faces to transmembrane segment 1 (TM1) mimics the "locked" signalling phenotypes of the wild-type receptor. The results are consistent with a model in which TM2 rotates in the plane of the lipid bilayer, and the rotation becomes locked at one face of the alpha-helix in the presence of attractant and at another face in the presence of repellent. This dynamic movement of the transmembrane domain may be a common signalling mechanism of homologous membrane receptor molecules such as the insulin receptor. Random-cassette mutagenesis and disulfide cross-linking provide powerful strategies for examining the structure and function of transmembrane segments.

Amino Acid Sequence↗
PubMed1994

Lambda foo: a lambda phage vector for the expression of foreign proteins.

This work describes a lambda phage expression system, lambda foo, that produces foreign proteins fused to the surface of the virus particle. The lambda foo vector has multiple cloning sites for the insertion of a foreign DNA fragment and color selection for recombinants. Foreign proteins are fused to the C terminus of a truncated phage tail protein, pV, by a peptide linker. Conditional chain termination allows the assembly and fusion of multisubunit proteins. We have attached the complete Escherichia coli beta-galactosidase and the plant Bauhinia purpurea agglutinin by cloning their genes into the vector. The constructs express functionally active proteins on the phage particle surface and have been purified by affinity chromatography with an antibody for beta-galactosidase and a mucin as a ligand for Bauhinia purpurea agglutinin.

Amino Acid Sequence↗
PubMed1993

A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant.

A bacterial expression plasmid, pBT7, based on a bacteriophage T7 RNA polymerase/promoter system, has been devised for positive color selection of clones containing an insert. The T7 gene 10 promoter and its N-terminal portion, 89 nucleotides in length, have been amplified by the polymerase chain reaction and cloned into the multiple cloning site of plasmid pUC18 in the opposite direction to lacZ'. This insertion does not inactivate the lacZ alpha-peptide activity. Hence, bacteria carrying pBT7 without an insert form blue colonies on an indicator plate, while bacteria carrying recombinant clones with an insert appear as white colonies. Using pBT7, part of the Caenorhabditis elegans unc-13 gene product has been overproduced at a high level, approaching 50% of total Escherichia coli protein.

Animals↗
PubMed1992

The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor.

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

Amino Acid Sequence↗
PubMed1992

A selective lambda phage cloning vector with automatic excision of the insert in a plasmid.

A bacteriophage lambda cloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (1) It has a unique BamHI site engineered into the lambda gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi- phenotype). (2) When the recombinant phage infects a Cre-producing E. coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids. The vector, lambda MGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.

Amino Acid Sequence↗
PubMed1991

A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans.

Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system.

Amino Acid Sequence↗
PubMed1989

Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family.

The sequences of three myosin heavy chain (MHC) genes from Caenorhabditis elegans, myo-1, 2 and 3, are presented. These genes, together with unc-54, comprise the entire nematode sacromeric MHC family. Comparison of nematode MHC sequences and sarcomeric, smooth and non-muscle MHCs from other organisms highlights conserved sequence features of the MHC rod believed to be important for thick filament assembly. These include: conservation of sequence differences between individual 28 amino acid repeats; invariant placements of large aromatic residues, such as tryptophan, in the rod sequences; conservation of "weak spots" in the hydrophobic seam; and conservation of non-uniform charge distributions along the length of the rod. The rod sequences of the body wall isoforms A and B are more closely related to each other than to the pharyngeal isoforms C and D, suggesting that structural constraints have been imposed by their location within the same thick filament. We have also identified the major transcriptional start site for gene unc-54. Surprisingly, there are no TATA or other known transcription factor elements immediately upstream from the unc-54 start site, or in the upstream regions of the other genes of the C. elegans MHC gene family.

Amino Acid Sequence↗
PubMed1989

Myosin heavy chain gene amplification as a suppressor mutation in Caenorhabditis elegans.

In the nematode, Caenorhabditis elegans, the body wall muscles contain paramyosin and two different types of myosin heavy chain, MHC A and MHC B. In mutants that do not express MHC B or that express defective paramyosin, muscle structure is disrupted and movement is impaired. Second site mutations in the sup-3 locus partially reverse these defects and are correlated with a 2- to 3-fold increase in the accumulation of the MHC A isoform. The sup-3 mutations occur at a high frequency (10(-4] after ethyl methanesulfonate (EMS) mutagenesis. This is comparable to the average EMS-induced mutation rate per gene in C. elegans. In this paper we show that the sup-3 mutation is an amplification of the structural gene for the MHC A protein, myo-3. We employed genomic Southern hybridization with MHC gene-specific probes in order to measure the copy number of the myo-3 gene relative to that of the MHC B gene, unc-54. We have identified the putative amplification junctions for these sup-3 alleles using a set of cosmid clones which encompass myo-3 region. Although it has been suggested that gene amplification plays an important role in evolution, there are few known cases of gene amplification in the germ line cells of multicellular organisms. The results shown here provide a clear example of a heritable gene amplification event that occurs at a high frequency in the germ line. Similar events may thus represent the initial event in the evolution of new function and in the formation of multigene families.

Animals↗
PubMed1988

Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60,000 molecular mass. The refolded PBP3** bound benzylpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain.

Acyltransferases↗
PubMed1983

On the process of cellular division in Escherichia coli: nucleotide sequence of the gene for penicillin-binding protein 3.

We determined the nucleotide sequence of a DNA fragment containing the ftsI gene coding for the penicillin-binding protein 3 (PBP-3), an indispensable enzyme for cell division of Escherichia coli. The entire ftsI gene was within the 2.8 kilobase PvuII fragment derived from the chromosomal segment on pLC26-6 (Nishimura et al. 1977). The coding region for PBP-3 was identified by comparison with the N-terminal amino acid sequence of in vitro synthesized PBP-3. The structural gene for ftsI consisted of 1,764 base-pairs coding for a 588 amino acid residue-polypeptide with a molecular weight of 63,850. PBP-3 synthesized in vitro showed a lower mobility in SDS-gel electrophoresis than that of the authentic PBP-3, suggesting that the primary translation product of the ftsI gene may be processed to yield mature PBP-3.

Bacterial Proteins↗
PubMed1981

Fluorospectrometric studies on neothramycin and its reaction with DNA.

The characteristics of neothramycin and its reaction mechanism with DNA were studied by fluorescence spectroscopy. The fluorescence intensity of neothramycin is enhanced by the reaction with DNA. The reaction rate of the drug and DNA is rather slow; and is dependent upon both DNA and drug concentrations, and is stimulated by hydrogen ion. Analysis of the equilibrium reaction suggested that neothramycin possesses about one binding site per 2-3 base-pairs with homogeneous affinity and association constant of 4.7 x 10(3) M-1; and no significant mutual interference of the binding sites seems to exist. From the results of neothramycin's interaction with various polynucleotides, it was suggested that a guanine base and a double helix conformation of DNA are required for binding the antibiotic. The current results and previous ones, concerning neothramycin-2'-deoxyguanosine adduct formation, suggested that the antibiotic reacts with 2-amino group of guanine base of DNA by dehydration in the manner of a bimolecular (Sn2ca) equilibrium reaction.

Antibiotics, Antineoplastic↗
PubMed1978

Isolation of a mutant of Escherichia coli lacking penicillin-sensitive D-alanine carboxypeptidase IA.

A mutant of Escherichia coli that is deficient in D-alanine carboxypeptidase IA has been isolated. The enzyme is membrane bound and moderately sensitive to penicillin. It catalyzes in vitro both D-alanine carboxypeptidase and transpeptidase reactions. Being able to synthesize crosslinked peptidoglycan both in vivo and in vitro despite the absence of enzyme activity, the newly isolated mutant grew normally under a wide range of growth conditions. Therefore, this enzyme, like D-alanine carboxypeptidase IB, is not required for normal peptidoglycan synthesis in E. coli. The defect in the activity of D-alanine carboxypeptidase IA in the mutant however was not associated with disappearance of penicillin-binding proteins 5 and 6 (which have been shown to be D-alanine carboxypeptidase IA) or any of the other protein bands that bind [14C]penicillin G. Genetic mapping studies showed that the mutation (dacA) is located close to leuS(13.7 min) on the E. coli chromosome map. Double mutants (dacA dacB) that are deficient in both D-alanine carboxypeptidases IA and IB were obtained. These double mutants also were found to grow normally and to catalyze normal formation of crosslinked peptidoglycan.

Carboxypeptidases↗