Toshikazu Fukui

Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF)

cDNA clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) were isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence reveals the primary structure of the MCAF precursor to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. The amino acid sequence of MCAF showed 25-55% homology with other members of an inducible cytokine family, including macrophage inflammatory protein and some putative polypeptide mediators known as JE, LD78, RANTES and TCA-3. This suggests that MCAF is a member of family of factors involved in immune and inflammatory responses.

Studies of nucleosides and nucleotides.: LVIII. Deamination of adenosine analogs with calf intestine adenosine deaminase☆

Several synthetic adeonosine analogs: 8-fluoro-, 8-azido-, 8-iodo-, 8-methylthioadenosine; 8-bromo-2′-deoxyadenosine, 8-bromoxylofuranosyladenine, 5′-benzoly-8-bromoadenosine; 8,2′-S-, 8,2′-O-, 8,2′-NH-, 8,2′-N-CH3-, 8,3′,-S-, 8,3′-O-, 8,5′-S- and 8,5′O-cycloadenosine; 1-deaza- and 3-deazaadenosine, as well as tubercidine (7-deazaadenosine), were tested as substrates of calf intestine adenosine deaminase. It was found that the adenine base of adenosine should be in the range φrmCN = 0–120° (anti to syn-anti) and 8-fluoroadenosine was hydroylzed very slowly. The purine base should have N1, N3 or N7 atoms for the hydrolysis and only 1-deazaadenosine revealed an inhibitory effect toward the hydrolysis of adenosine. 5′-OH group should be in the position of S-configuration and must not be substituted.

Reversed-phase ion-pair chromatography of oligodeoxyribonucleotides

The separation of large oligodeoxyribonucleotides, synthesized chemically and subsequently deblocked, was carried out by reversed-phase ion-pair chromatography (RP-IPC) with a linear gradient of acetonitrile concentration. It was found that tetrabutylammonium phosphate is suitable as an ion-pairing reagent and produces a linear relationship between the base numbers of the samples and their elution volumes. It was also verified that various factors effective in reversed-phase chromatography, such as temperature, end-capping of the columns, differences in the type of C18 alkylating reagents and in the base silica, and the pore size of the base silica have little effect on the resulting separation by RP-IPC.

Behaviour of single-stranded oligodeoxyribonucleotides on a deae-5pw anion-exchange column

Abstract The separation of large, single-stranded oligodeoxyribonucleotides, prepared by chemical synthesis, was carried out on a DEAE-5PW anion-exchange column with eluents containing only volatile salts. It was found that gradient elution is essential and that a high resolution can be achieved at elevated temperatures ( ca. 50°C). Also, neutral pH of the eluents was found to be desirable for the separation. Under optimum conditions, a linear relationship between the elution volumes and the number of bases in the substrates was observed.

A reversed-phase liquid chromatographic columns for the analysis of single-stranded oligo-DNAs

SummaryA reversed-phase liquid chromatographic (RPLC) column, with which large oligodeoxynucleotides (oligo-DNAs) can be separated in well-resolved peaks, has been developed. The column contains only specially selected silica bonded with monochlorodimethyloctadecylsilane. The carbon content of the column packing is the predominant factor to achieve good results. With this column several mixtures containing different large oligo-DNAs could be separated with excellent resolution.

Cloning of the Escherichia coli gene for the stringent starvation protein

SummaryIn order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5′-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).

Separation of single-stranded oligonucleotides on polyvinyl alcohol gel column

SummarySeparation mechanisms for single-stranded oligodeoxyribo-or oligoribonucleic acid fragments were explored on an Asahipak polyvinyl alcohol gel column (GS-320) by use of sequential isomers of such molecules. Substrates having different base numbers were found to be separated by size-exclusion chromatography while those having the same numbers with different base sequences were isolated by use of the reversed-phase mode. By using those dual modes, a limit for the separation of the samples was found to arise because one mode shifted the peaks of the substrates in the sense opposite to the shift resulting from the other mode and it was found that when substrates had less than nine bases, the solutes eluted separately.

Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid) . poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine . uracil basepair has been found to exchange at a measurably slow rate, 0.023 s-1 (at 0 degrees C), which is, however, much greater than that for a double-helix with the Watson-Crick type A . U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU) . poly(rA) . poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U . A. U base triplet have been found to exchange at a relatively slow rate, 0.0116 s-1 (at 0 degrees C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U. A. U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.