Biomedical subjects

E Ohtsubo

Publications and source records attributed to E Ohtsubo.

At least 73 records · Page 4Linked to original sources

PubMed1993

Classification and relationships of rice strains with AA genome by identification of transposable elements at nine loci.

We analyzed the presence of p-SINE1 members at five loci in the rice strains belonging to seven species with AA genome in the Oryza genus by the methods including polymerase chain reaction (PCR). Four p-SINE1 members (p-SINE1-r3, r4, r5 and r7) were present at the corresponding loci in all the strains examined. One member (p-SINE1-r6) was, however, not present at the corresponding locus in most of the African strains of O. glaberrima and O. barthii, but was in the other strains. The PCR-amplified fragments containing p-SINE1-r4 in many strains were found to be larger due to insertion of either one of two transposable elements, named Tnr2 and Ret1, within or near p-SINE1-r4, respectively: Tnr2 is 157 bp in length with terminal inverted repeat sequences of about 56 bp; Ret1 is only 13 bp in length with a T stretch at its end. Tnr2 was not present in the corresponding locus in all the strains belonging to O. sativa Japonica and in some strains of O. rufipogon and O. longistaminata, while Ret1 was present only in the two strains of O. longistaminata. These results and previous ones obtained from the analysis of the other two p-SINE1 members (p-SINE1-r1 and r2) in the Wx gene indicate that the elements, such as p-SINE1-r6, Tnr2, Ret1 and p-SINE1-r2, have been inserted into the respective loci during divergence of the rice species with AA genome. The patterns for the presence and absence of the transposable elements at the respective loci enabled us to classify the rice strains with AA genome into ten groups and to infer their relationships.

Base Sequence↗

PubMed1992

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.

Amino Acid Sequence↗

PubMed1992

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.

Adenine↗

PubMed1992

DNA sequences required for translational frameshifting in production of the transposase encoded by IS1.

The transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames (insA and B'-insB) by translational frameshifting, which occurs within a run of six adenines in the -1 direction. To determine the sequence essential for frameshifting, substitution mutations were introduced within the region containing the run of adenines and were examined for their effects on frameshifting. Substitutions at each of three (2nd, 3rd and 4th) adenine residues in the run, which are recognized by tRNA(Lys) reading insA, caused serious defects in frameshifting, showing that the three adenine residues are essential for frameshifting. The effects of substitution mutations introduced in the region flanking the run of adenines and in the secondary structures located downstream were, however, small, indicating that such a region and structures are not essential for frameshifting. Deletion of a region containing the termination codon of insA caused a decrease in beta-galactosidase activity specified by the lacZ fusion plasmid in frame with B'-insB. Exchange of the wild-type termination codon of insA for a different one or introduction of an additional termination codon in the region upstream of the native termination codon caused an increase in beta-galactosidase activity, indicating that the termination codon in insA affects the efficiency of frameshifting.

Amino Acid Sequence↗

PubMed1992

The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth.

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.

Bacterial Proteins↗

PubMed1992

Characterization of a plant SINE, p-SINE1, in rice genomes.

We have previously found that a short interspersed element (SINE), named p-SINE1, is present in the Waxy gene of Oryza sativa in two copies. Here, we cloned five members of p-SINE1 located at other loci in O. sativa and determined their nucleotide sequences. These sequences had a T-rich pyrimidine tract at their defined 3' end and were flanked by direct repeats of a sequence of mostly 14-15 bp long like p-SINE1s in the Waxy gene. The consensus sequence derived from total seven members of p-SINE1 was 123 bp in length and had an internal promoter region for RNA polymerase III. The 5'-half region of the sequence was partially homologous to the tRNA-related block of rabbit C family, one of SINEs in the animal system. Two of the seven p-SINE1 members were not present in the corresponding loci in African rice, Oryza glaberrima, and may thus be available for classification of some rice strains. Comparison of the nucleotide sequences of the Waxy gene between O. sativa and O. glaberrima showed that base substitutions have frequently occurred in a p-SINE1 member (p-SINE1-r1) and a transposable element Tnr1 also present in the Waxy gene, suggesting that these elements, which appear as repetitive sequences in the rice chromosome, tend to acquire base substitutions at a higher frequency than do unique sequences.

Base Sequence↗

PubMed1991

Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.

Bacteriocin Plasmids↗

PubMed1991

Functional analysis of the two domains in the terminal inverted repeat sequence required for transposition of Tn3.

Bacterial transposon Tn3 has a 38-bp terminal inverted repeat (IR) sequence. The IR sequence has been divided into two domains, A and B, of which domain B is bound by transposase, and domain A is not Here, we defined the two domains more precisely by constructing three IR mutants with a 2-bp substitution at relevant sites within the IR sequence, followed by examination of the binding of transposase to the fragments containing these IR mutants: domain A was located at bp 1-11, whereas domain B was at bp 12-38. To see if the two domains in the IR are functionally distinct, we constructed mini-Tn3 derivatives flanked by two IRs with various 2-bp substitutions within domain A or B, and analyzed their ability to mediate cointegration. The mini-Tn3 derivatives flanked by IR(A+ B+) and IR(A- B+) [or IR(A+ B-)] and those flanked by IR(A-B+) and IR(A+ B-) mediate cointegration more efficiently than the mini-Tn3 derivatives flanked by two IR(A- B+)s or by two IR(A+ B-)s. These results and others presented here indicate that the two domains of IR are functionally distinct in promoting cointegration.

Base Sequence↗

PubMed1991

Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100.

We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100. The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein. This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100. The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick. The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA.

Bacterial Proteins↗

PubMed1991

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.

Amino Acid Sequence↗

PubMed1991

Preferential transposition of an IS630-associated composite transposon to TA in the 5'-CTAG-3' sequence.

A composite transposon, Tn4731, associated with IS630 has been shown to transpose preferentially to 5'-TA-3' sequences that are located at two sites in a rho-dependent transcription terminator in plasmid ColE1 in Escherichia coli (T. Tenzen, S. Matsutani, and E. Ohtsubo, J. Bacteriol. 172:3830-3836, 1990). Here we demonstrated that Tn4731 preferentially transposes to TA sequences at four sites in plasmid pUC118 and its derivatives: the TA sequence (hot spot I) in the intergenic region of phage M13 within the pUC sequence, the TA sequence (hot spot II) in the XbaI site in multiple cloning sites of the lacZ coding region, the TA sequence (hot spot III) in a spacer region flanked by inverted repeat sequences of a transcription terminator located downstream of the bla gene, and the TA sequence (hot spot IV) in the middle of bla. Transposition of Tn4731 to hot spot III was found not to require the inverted repeats in the terminator. Transposition of Tn4731 to hot spot II, which is located immediately downstream of the lacZ promoter, was not affected by mutations introduced into the promoter. There appear to be no particular sequences important for transposition of Tn4731 around each of the hot spots, except a palindromic sequence, 5'-CTAG-3', that contains the target sequence. Mutations introduced within the CTAG sequence at a hot spot inhibited Tn4731 from transposing to it, indicating that the CTAG sequence is responsible for the preferential transposition of Tn4731.

Base Composition↗

PubMed1991

Specific DNA binding of the TraM protein to the oriT region of plasmid R100.

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.

Amino Acid Sequence↗

PubMed1991

Organization of DNA sequences highly repeated in tandem in rice genomes.

Digestion of the total genomic DNA from rice Oryza sativa L. cv. C5924 with EcoRI generated an intense band of a DNA fragment of about 0.36 kb long. The DNA fragment cloned into pUC19 was used to hybridize with the total rice genomic DNA partially digested with EcoRI. A ladder of bands of DNA fragments with multiplied length of 0.36 kb was observed, demonstrating that this sequence occurs in tandem in the genome. The copy number of the sequence estimated by dot blot hybridization analysis was 2000-3000 copies per haploid genome from callus or seedling of C5924. This sequence was present in other O. sativa cultivars, such as Sasanishiki in 700-900 copies, Koshihikari in 3400-4300, and Nipponbare in 4600-6000 copies. Another rice species, O. glaberrima, also had this sequence in 540-680 copies, but four lines of foxtail millet had none. The DNA fragments containing the repeated sequences in Nipponbare were then cloned into lambda EMBL3, and sequences of nine units consecutively repeated and an AT-rich sequence connected with them in a phage clone could be determined. Each repeating unit showed sequence divergency mostly by substitution of bases in a range from 3% to 7%, when compared with a 355-bp consensus sequence. Analyses of the substituted bases indicate that these are due to spontaneous mutations which occurred at random, after reiteration of a unit sequence by unequal crossing over events. Gene conversion within the repeated sequences might have further diversified their sequences.

Base Sequence↗

PubMed1991

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.

Amino Acid Sequence↗

PubMed1990

In vitro transposition of transposon Tn3.

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.

Ampicillin Resistance↗

PubMed1990

Differential scanning calorimetric study of the effect of intercalators and other kinds of DNA-binding drugs on the stepwise melting of plasmid DNA.

The effect of intercalating drugs (the anthracycline group of antibiotics, ethidium bromide, actinomycin D) on stepwise melting of DNA was studied by differential scanning calorimetry (DSC). The DSC DNA melting profile of plasmid pJL3-TB5 DNA (5277 base-pairs in length) consists of seven peaks, and all the intercalators caused shifting of these peaks, particularly those formed at the high temperature ranges, to the higher temperature ranges in a characteristic manner depending upon the binding strength of the drug. The analysis of the anthracycline group of antibiotics, such as aclacinomycin A, daunomycin, adriamycin and pyrarubicin, indicates that the difference in binding is due to the sugar moiety at position O-7 of the chromophore in these antibiotics. Analysis on the basis of the helix-coil transition theory suggests that the anthracycline group of antibiotics interact preferentially with the 5'-CG-3' sequences. The effect of various DNA-binding drugs other than intercalators on stepwise melting of DNA was then studied by DSC. The representative drugs examined were distamycin A, peplomycin, cis-dichlorodiamine-platinum(II) (cis-DDP or cis-Platin) and mitomycin C, which differ in their mode of interaction with DNA; namely, minor groove binding, strand cleavage and intrastrand or interstrand cross-linking. Distamycin A caused shifting of the DSC peaks at the low temperature ranges to a higher temperature range, whereas peplomycin and cis-DDP caused shifting of all the DSC peaks to form a broad peak at a lower temperature range, suggesting that the DSC DNA melting profiles are affected in a characteristic manner depending upon the interaction mode of the drug.

Antibiotics, Antineoplastic↗

PubMed1990

Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes.

The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.

Amino Acid Sequence↗

PubMed1990

Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5.

We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110. Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them. We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome. Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E. coli K-12 strain, JE5519. While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5. Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present. In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E. coli K-12 derivatives.

Bacteriophage lambda↗