Biomedical subjects

T Komano

Publications and source records attributed to T Komano.

At least 109 records · Page 6Linked to original sources

PubMed1991

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.

Base Sequence↗

PubMed1991

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli.

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.

Amino Acid Sequence↗

PubMed1991

Structural analysis of repetitive DNA sequences in the goat growth hormone gene region.

Two clones that contain goat growth hormone (gGH) genes were isolated from goat genomic library using goat growth hormone cDNA as a probe. One clone CgGH contained gGH1 gene, and another clone, EgGH, contained gGH2 and gGH3 genes. The clone EgGH contained 491 bp inserted sequence, just upstream of the gGH3 gene, which was not present in the clone CgGH. From the sequencing data of the flanking regions of these gGH genes, short interspersed repetitive sequences (SINES) were found. Four SINES's existed in the CgGH clone and eight SINES's existed in the EgGH clone. The number of the repetitive DNA sequences in the goat genome was estimated about 10(2) copies from the analysis of the reassociation rate. As the number of gGH genes (gGH1, gGH2, and gGH3) per genome was different among individual goats, DNA fragments containing gGH1 gene, and that containing gGH2 and gGH3 genes, were estimated to be allelic on the goat chromosome.

Animals↗

PubMed1991

Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein.

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Expression of human P-glycoprotein in yeast cells--effects of membrane component sterols on the activity of P-glycoprotein.

A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis.

45 mutations (insertion, deletion and base substitution) of the G4 Goric were tested for their functional activity in M13 and R199 in vivo. The critical mutants were also assayed for their ability to synthesize pRNA in vitro using SSB and primase. The results demonstrate that the secondary structure and spacing of stem-loops I and III are essential for Goric activity and that the 5'-CTG-3' sequence flanking stem-loop I is essential for initiation of pRNA synthesis.

Bacteriophages↗

PubMed1990

Physical and genetic analyses of IncI2 plasmid R721: evidence for the presence of shufflon.

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.

Conjugation, Genetic↗

PubMed1990

Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.

Chromosome Deletion↗

PubMed1990

The effects of metal ions on the DNA damage induced by hydrogen peroxide.

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.

Bacteriophage phi X 174↗

PubMed1989

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase.

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.

Base Sequence↗

PubMed1989

Distinct functional contributions of three potential secondary structures in the phage G4 origin of complementary DNA strand synthesis.

Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I, and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.

Base Sequence↗

PubMed1989

RepB' is required in trans for the two single-strand DNA initiation signals in oriV of plasmid RSF1010.

We have shown previously [Honda et al., Gene 68 (1988) 221-228] that the oriV region of the broad-host-range plasmid RSF1010 contained two single-strand DNA initiation signals (ssi) which have RSF1010-specific properties since they required one or more factors provided in trans by RSF1010 for their functional activities. We demonstrate here, by deletion analysis, that repB', one of the genes essential for the vegetative replication of RSF1010, produces a factor required for the function of ssi signals. It is conceivable that the RepB' protein and the two ssi signals, ssiA and ssiB, cooperatively compose plasmid-specific priming complexes which confer the broad-host-range property upon RSF1010.

Bacterial Proteins↗

PubMed1989

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance.

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Identification of single-strand initiation signals in the terC region of the Escherichia coli chromosome.

On the basis of clear-plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome. At least two single-strand initiation signals were identified from the terC region. The nucleotide sequences of these two signals were determined. Sequence homologies, variations of the consensus of n' protein recognition sites, 5'-GAAGCGG-3', were found within these signals. A novel conserved sequence was also found within these signals. Their initiation activities were measured both by the infection growth assay and by the ability to convert the single-stranded DNA to the duplex replicative form DNA in vivo.

Bacteriophage phi X 174↗

PubMed1989

Mutational analysis of an ssi region carried by plasmid pACYC184.

To investigate the functional contribution of some structural components of the signal that directs single-stranded initiation of DNA replication (ssi signal) carried by a 119-nt segment of plasmid pACYC184 (Bahk et al., 1988), we constructed mutants carrying one-base substitutions and insertions using oligodeoxyribonucleotide (oligo) directed mutagenesis. Two one-base substitution mutants were obtained. The mutants, M13 delta lac 184/Sp and M13 delta lac 184/Ev, carried an SplI site and an EcoRV site, respectively, created by base substitution. Three kinds of synthetic oligos, that is, a 10-bp EcoRI linker, an 8-bp ScaI linker and an 8-bp SmaI linker, were inserted into the SplI site of M13 delta lac 184/Sp, and into the EcoRV site of M13 delta lac 184/Ev. The SSI activity of each mutant examined indicated that the one-base substitutions had different effects on the SSI functions of the altered ssi signals. This fact suggests that some structural components within the 119-bp region make distinct contributions to the SSI function. Moreover, when the three kinds of synthetic linkers were inserted into the mutants M13 delta lac 184/Sp and M13 delta lac 184/Ev, each of the insertion mutations affected the rate of conversion of ss DNA to RFI in vivo and the growth of the recombinant phages in a distinct manner. Judging from the above results, the base composition and the length of a certain specific site were crucial for maintenance of the SSI functional activity, and structural components of the ssi signal contributed distinctly to the SSI function.

Bacteriophages↗

PubMed1989

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli.

The glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87,000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5'-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene.

Amino Acid Sequence↗

PubMed1989

Cloning and nucleotide sequence of the ColIb shufflon.

The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.

Base Sequence↗