Cloning and sequencing of the rabbit gene encoding T-cell costimulatory molecules

The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D49841 (RCD28), D49844 (RCTLA-4), D49842 (RCD80), and D49843 (RCD86)     

Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.     

Intraspecific brood-mixing of the cichlid fishPerissodus microlepis in Lake Tanganyika

Synopsis The frequency and origin of intraspecific brood-mixing in the biparental cichlid fishPerissodus microlepis were investigated by the cohort analysis of schooling young and the underwater observation of guarding parents. The cohort analysis showed that brood-mixing started from the early guarding state when the young were smaller than 10 mm standard length and nearly all schools of young larger than 16 mm contained alien young from up to 6 broods. Brood farming-out of this fish, which was originally proposed to be a way adopted only by a deserted parent, was performed also by paired parents. We suggest that brood-mixing inP. microlepis is attributed mainly to brood farming-out by paired and deserted parents.     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Synthesis of desmosterol and epidesmosterol from hyodeoxycholic acid

A new synthesis of desmosterol was described using hyodeoxycholic acid (3,6-dihydroxy-5β-cholanic acid) as a starting material. Epidesmosterol (3-hydroxycholesta-5,24-diene) was also synthesized for the first time from the same starting material.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

The augmentative effect of a protein-bound polysaccharide (PSK) on tumoricidal activity of the soluble factor produced by a streptococcal preparation (OK-432)-activated macrophages

The enhancement of antitumor activities of the tumoricidal soluble factor (SF) from a streptococcal preparation (OK-432)-activated macrophages by the pretreatment with a protein-bound polysaccharide (PSK) was investigated in tumor-bearing mice.Two-step stimulations with OK-432 atin vivo priming andin vitro eliciting were required for the production of the tumoricidal SF by macrophages, and the tumoricidal activity of the SF apparently correlated with the uptake of OK-432 by macrophages at priming phase.Tumoricidal activity of the SF from OK-432-activated macrophages in proteose-peptone (P-P)-pretreated mice significantly decreased with the development of the tumor, whereas in PSK-pretreated mice did not. Pretreatment of tumor-bearing mice with PSK saved a decrease in the macrophages carrying Ia^k or asialo GM1 antigens and an increase in wheat germ agglutinin (WGA) receptors. Furthermore, the uptake of OK-432 by macrophages at priming phase was enhanced. The tumoricidal activity of the SF from OK-432-activated macrophages was augmented.Thus, PSK may restore the depressed functions of macrophages, and the combination therapy with PSK and OK-432 may be effective to enhance the production of tumoricidal SF in tumor-bearing mice.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.