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Y Kohara

Publications and source records attributed to Y Kohara.

At least 73 records · Page 4Linked to original sources
PubMed1989

Construction of an ordered cosmid collection of the Escherichia coli K-12 W3110 chromosome.

A cosmid library of the Escherichia coli K-12 W3110 chromosome was constructed in which clones were assigned to locations on the chromosome map by hybridization and genetic marker complementation tests. Approximately 70% of the genome was represented by this library. The identified clones can be maintained in the homologous system and would facilitate genetic studies of E. coli.

Chromosome Mapping↗
PubMed1988

Genome linking with yeast artificial chromosomes.

The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.

Animals↗
PubMed1988

Insertion sequence IS5 contains a sharply curved DNA structure at its terminus.

It was demonstrated that insertion sequence IS5 contains a sequence-directed bent (sharply curved) DNA structure at its terminus, close to one of its 16 bp terminal repeats. The minimal number of copies of IS5 related sequences and the locations of the latter on the Escherichia coli K12 W3110 chromosome were determined. Evidence is presented of the occurrence of IS5 mediated translocation and duplication of a large DNA segment on the E. coli chromosome.

Bacteriophage lambda↗
PubMed1988

Change in priming sites for discontinuous DNA synthesis between the monomeric and concatemeric stages of phage T7 replication.

We have analyzed the transition sites between primer RNA and DNA in a 589 bp segment of the bacteriophage T7 genome. In the monomeric replication stage, RNA-DNA transition sites are predominantly on the light (L) strand (with 5'----3' polarity on the genetic map) but rarely on the heavy (H) strand, indicating that replication proceeds semidiscontinuously with the H and L strands corresponding to the leading and lagging strands, respectively. The direction of replication is that expected from the position of the primary origin and also indicates that secondary origins are seldom if ever used. In the concatemeric stage of replication, RNA-DNA transition sites are instead distributed on both strands of the segment with equally high frequency, showing that initiation occurs within the concatemeric molecule per se and by a different mechanism.

Base Sequence↗
PubMed1988

Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches.

The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome.

Amino Acid Sequence↗
PubMed1987

The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library.

Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms.

Chromosome Mapping↗
PubMed1987

Relative roles of T7 RNA polymerase and gene 4 primase for the initiation of T7 phage DNA replication in vivo.

Initiation sites of T7 phage DNA replication in the presence and absence of T7 phage gene 4 primase have been analyzed by using Escherichia coli cells infected with T7 phage amber mutants, T73,6 and T73,4,6, respectively. Restriction analysis of the [3H]thymidine-labeled DNA, synthesized by the T73,4,6 phage-infected cells in the presence of 2',3'-dideoxy-3'-azidothymidine, has shown that only the light (L) strand of T7 DNA has been synthesized from the primary origin area to the right. Transition sites from RNA to DNA have been located precisely in the primary origin region of the T7 phage genome. In the gene 4- condition, greater than 20 transition sites have been detected only in the L strand. They scattered widely downstream from the phi 1.1 promoters and mostly downstream from the phi 1.3 promoter. The same transition sites have been detected in the gene 4+ condition, suggesting that the transcripts started from these promoters are used as primers of the rightward L-strand DNA synthesis in the gene 4+ condition. In addition, many heavy (H)- and L-strand transition sites have been detected at gene 4 primase sites in the gene 4+ condition. The relative roles of T7 phage RNA polymerase and primase at the primary origin have been discussed.

Base Sequence↗
PubMed1985

The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome.

RNA-linked DNA molecules were obtained from E. coli dnaCts cells synchronously initiating a new round of chromosome replication. The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region. In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region. In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region. The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC. Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length. Properties of the transition sites were discussed.

Base Sequence↗
PubMed1983

[Giant left atrium in mitral valve disease: a new plication procedure to relieve the compressions of left ventricular wall left bronchus and right lung].

The giant left atrium associated with mitral valve disease frequently produces postoperative hazardness in relation to hemodynamic and respiratory management. We have defined the most serious disorders induced by the presence of giant left atrium in three categories as follows. First, hemodynamic disturbance by the compression of left ventricular wall by downward extension of left atrium (type I), secondly, respiratory disturbance yielded by the compression of left main bronchus by upward extension of left atrium (type II), thirdly, compression of right middle lobe by rightward extension of left atrium (type III). A new procedure of para-annular, superior and right-side plication methods were derived as the procedure to relieve those compressions induced by giant left atrium. Up to the present, 47 patients with giant left atrium underwent surgery, twelve of valvular procedure only and thirty-five of valvular as well as plication procedure. The incidence of postoperative low output syndrome and respiratory failure were evaluated. The plication procedure showed marked decrease in the incidence of low output syndrome and respiratory failure postoperatively, eventual significant decrease in mortality rate. We conclude that plication procedure is very effective for the treatment of compression in the presence of giant left. atrium.

Cardiac Output, Low↗
PubMed1981

Initiation sites for discontinuous DNA synthesis of bacteriophage T7.

We have previously shown that the discontinuous replication of bacteriophage T7 DNA is primed by tetraribonucleotides (major component) or pentaribonucleotides. Both tetramers and pentamers start with pppA-C and are rich in A and C at the third and fourth nucleotides. In this study, the sites of transition from primer RNA to DNA in vivo have been located on a 340-nucleotide segment of the H strand of the T7 genome by 32P-labeling in vitro of the 5'-hydroxyl ends of DNA resulting from alkaline hydrolysis of RNA-linked T7 DNA fragments. Five strong transition sites were detected with a common sequence 5'-G-A-C-N1-N2-N3-N4-3', in which N1 was either C or A, N2 ws A, C, or G, and either N3 or N4 was the nucleotide for the switchover to DNA synthesis. We conclude that the complementary sequence 3'-C-T-G-G/T-N'2-(N'3)-5' in the template strand is the most frequently used signal for synthesis of primer RNA. Whereas primer-RNA synthesis starts at a precisely defined nucleotide, the transition to DNA synthesis varies within two nucleotides. Because the observed signal sequence would be present on a statistical basis once per 128 nucleotides, only about 10% of the existing signals are used for primer synthesis in each round of replication so that nascent fragments 1000-2000 long result. This provides an unexpected flexibility for RNA priming of DNA synthesis.

Base Sequence↗