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Biomedical subjects

K Sakaguchi

Publications and source records attributed to K Sakaguchi.

At least 469 records · Page 26Linked to original sources
PubMed1979

Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp- phenotype to Trp+. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and beta subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.

Anthranilate Synthase↗
PubMed1979

Addition of short guanylyl blocks to oligonucleotide primers with a thermophilic polynucleotide phosphorylase. Its application to the synthesis of oligonucleotides containing guanylyl residues.

Polynucleotide phosphorylase from Thermus thermophilus catalyzed the addition of short guanylyl blocks from GDP to the 3'-hydroxyl termini of oligonucleotide primers at low temperature in a simple reaction mixture. Polyguanylic acid formation was inhibited at 37 degrees C, but the addition of one or two guanylyl residues to oligonucleotide primers proceeded in high yields. The reaction was applied to the synthesis of oligonucleotides containing guanylyl residues at the 3'-end. Using (Ap)2A and (Up)2U as primers, (Ap)3G, (Ap)3GpG, and (Up)3G were synthesized in yields of 25--52%. (Ap)2GpG was synthesized from ApA and GDP in a yield of 13%.

Guanine↗
PubMed1978

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu+ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B.leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu+. However, B. subtilis ilvB and ilvc auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. beta-Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.

2-Isopropylmalate Synthase↗
PubMed1978

Addition of mononucleotides to oligoribonucleotide acceptors with T4 RNA ligase.

RNA ligase induced by bacteriophage T4 catalyzed the addition of nucleoside 5',3'-diphosphates to oligoribonucleotide acceptors in the presence of ATP. The reactions proceeded in equimolar concentrations of donors and acceptors. Several oligonucleotides of defined sequence were synthesized by this method in yields of 28-68%. The enzyme required the phosphate ester at the 3' position of the donor molecule, nucleoside 5',2'-diphosphates being unable to serve as donors. Thymidine 5',3'-diphosphate was active as a donor for the enzyme. The ligation product, (Ap)2ApCp, was also obtained from the reaction of (Ap)2A and 5'-adenylylated cytidine 5',3'-diphosphate (A5'pp5'Cp) with RNA ligase in the absence of ATP. These results show that the minimal substrate for RNA ligase is a nucleoside 5',3'-diphosphate.

Adenosine Triphosphate↗
PubMed1978

Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.

The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.

Alkaline Phosphatase↗
PubMed1978

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria.

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.

DNA Restriction Enzymes↗
PubMed1978

RSF1010 plasmid as a potentially useful vector in Pseudomonas species.

RSF1010 plasmid DNA was introduced into Pseudomonas putida and P. aeruginosa cells and maintained stably, suggesting the potential usefulness of this plasmid as a vector in Pseudomonas species. The number of copies of RSF1010 was 43 per chromosome equivalent in P. putida cells.

DNA, Bacterial↗