Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of replication initiation site in Mycoplasma capricolum genome by two-dimensional gel-electrophoretic analysis.

The homolog of the dnaA gene, which has been reported to be present in the vicinity of the initiation site of replication in the genome of Mycoplasma capricolum (M.Miyata, L.Wang, and T.Fukumura, J. Bacteriol. 175: 655-660, 1993) was mapped precisely. A 9540-bp region containing the dnaA gene was cloned and the entire region was sequenced with the exception of a previously reported region of 2517 bp (Fujita, M.Q., Yoshikawa, H. and Ogasawara, N. Gene 93: 73-78, 1992). The organization of the 9540-bp region was compared with that of corresponding regions in other bacteria. The arrangement and directions of rnpA, rpmH, dnaA, dnaN were conserved, but no other open reading frames were found that were homologous to those that are commonly found around dnaA genes in other bacteria. The directions of movement of the replication fork around the dnaA gene were analyzed by neutral/alkaline two-dimensional gel electrophoresis. The forks developed in a 1569-bp region that consisted of the dnaA structural gene and its downstream non-coding region, and then they proceeded bidirectionally.     

Comparison of the conserved region in the dnaA gene from three mollicute species

Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria.     

Screening,Classification and Distribution of l-α-Amino-ε-caprolactam-hydrolyzing Yeasts

1. Screening of microorganisms which hydrolyzed l-α-amino-ε-caprolactam** into l-lysine from soil was undertaken to establish an enzymatic method of l-lysine production. Several yeast strains were isolated which grew on l-aminolactam and whose cells hydrolyzed it into l-lysine.

2. A taxonomic study on the yeasts isolated was performed and they were classified into three genera and species, Cryptococcus laurentii, Candida humicola and Trichosporon cutaneum.

3. Authentic yeast strains obtained from some organizations were screened for the ability to utilize and hydrolyze l-aminolactam and the above three genera and species were found to possess the ability.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.     

A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 x 10(8) to 1. 0 x 10(8) cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.     

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39 μm along the lateral direction and 1.3 μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94 K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90 K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790 nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800 nm spectral region.     

Biodiesel production using anionic ion-exchange resin as heterogeneous catalyst

The transesterification reactions of triolein with ethanol using various ion-exchange resin catalysts were conducted to produce ethyl oleate as a biodiesel. The anion-exchange resins exhibited much higher catalytic activities than the cation-exchange resin. The anion-exchange resin with a lower cross-linking density and a smaller particle size gave a high reaction rate as well as a high conversion. By combining the three-step regeneration method, the resin could be repeatedly used for the batch transesterification without any loss in the catalytic activity. A continuous transesterification reaction was carried out using an expanded bed reactor packed with the most active resin. The reactor system permitted the continuous production of ethyl oleate with a high conversion.