Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification.

The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322. The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose. The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant. The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product. The cls gene is thus most likely the structural gene coding for cardiolipin synthase. Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature. Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E. coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions.     

An investigation for the occurrence of C4 photosynthesis in the Cyperaceae from Australia

Two hundred and twenty species of 38 genera in the Cyperaceae from Australia were examined for the possible occurrence of the C₄ photosynthesis and the anatomical features of leaves and culms. The Kranz type of anatomy and the carbon isotope ratios typical of C₄ plants were found in 84 species in the following six genera of four tribes belonging to subfamily Cyperoideae:Bulbostylis, Crosslandia, andFimbristylis (Fimbristylideae);Lipocarpha (Lipocarpheae);Cyperus (Cypereae);Rhynchospora (Rhynchosporeae). The anatomical observation revealed that the C₄ species possessed any one of the three Kranz anatomical types found by previous investigators. It was suggested that in the Cyperaceae the C₄ syndrome evolved independently within several taxa of the subfamily. The relative distribution of C₃ and C₄ species of the Cyperaceae in Australia was investigated by use of floristic data. It was recognized that the C₄ species dominated in the northern part of the continent which was characterized by tropical and subtropical savannas and hot dry areas with summer rainfall, and the C₃ species in the southern part, which contained temperate areas and mediterranean climatic areas with winter rainfall.     

Respiration and avoidance reaction of a cyprinid fishPseudogobio esocinus under hypoxia

A cyprinid fish,Pseudogobio esocinus showed gradual bradycardia at oxygen saturation (%) of less than 29.7±4.6 (1.89±0.29 ml/l of oxygen concentration), surfacing at 14.7±1.3 (0.94±0.09ml/l), drastic decrease of oxygen consumption at less than 14.2±0.8 (0.91 ±0.06ml/l) and asphyxia at 9.7±1.4 (0.62±0.09ml/l). The fish avoided water having low oxygen saturation of less than 54.0± 5.4 (3.38±0.30ml/l), and markedly at less than 26.2±3.4 (1.62±0.16 ml/l).     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

Effect of Ferricyanide, Ferrocyanide and KCN on Growth and Flowering in the Short-Day Plant Lemna paucicostata 6746

Flowering in the short-day plant Lemna paucicostata 6746 canbe induced under continuous light by the addition of ferricyanie,ferrocyanide or KCN to M-sucrose medium. Each substance is nearly10 times more effective when the flasks are covered by glassbeakers than when cotton plugs are used. By contrast, when floweringis induced under continuous light by copper or by short-daytreatment, neither flowering nor growth are affected by whetherglass beakers or cotton plugs are used. Ferricyanide, ferrocyanideand KCN are also able to induce long-day flowering when theplants are grown on Msucrose medium in small beakers that areplaced in a covered storage dish that also contains a solutionof one of these compounds. Addition of a KOH trap to the storagedish completely blocks the flowering induced by these compounds.If [¹⁴C]ferrocyanide is added to the storage dish both the M-sucrosemedium and the plants contain significant amounts of radioactivity,the amount of radioactivity being proportional to the floweringresponse. These results indicate that ferricyanide, ferrocyanideand KCN break down to release HCN and that it is the HCN whichis responsible for inducing flowering in L. paucicostata 6746under continuous light. ¹Present address: Department of Biology, Osaka Kyoiku University,Ikeda, Osaka 563, Japan. ²Present address: Institute of Horticulture, The Volcani Center,P. O. B. 6, Bet-Dagan, Israel. (Received January 17, 1983; Accepted March 24, 1983)     

Flower choice in honeybees: Effects of food value and flower density

To test the effects of food value on the flower choice, individual honeybees (Apis mellifera) were offered a choice of 25 % sucrose solution (SS) and 1 of 6 different SSs, ranging from 5 % to 50 % SS, at either a low or a high flower density. Artificial flowers were filled with each SS. The honeybees showed a stronger preference for a concentrated SS to a diluted SS at a high than at a low flower density, and the degree of preference was positively correlated to the difference in the sucrose concentration between paired SSs. These foraging patterns were consistent with qualitative predictions from optimal foraging theory. Furthermore, it was found that experience in feeding on a concentrated SS lowered the foraging motivation for a diluted SS at the high flower density, but not at the low flower density. I discuss the effects of food density, food profitability and experience on the foraging behaviour of honeybees.     

Optimal sequence alignment allowing for long gaps

A new algorithm for optimal sequence alignment allowing for long insertions and deletions is developed. The algorithm requires O((L+C)MN) computational steps, O(LN) primary memory and O(MN) secondary memory storage, whereM andN(M≥N) are sequence lengths,L (typicallyL≤3) is the number of segment specifying the gap weighting function, andC is a constant. We have also modified our earlier traceback algorithm so that it finds all and only the optimal alignments in a compact form of a directed graph. The current versions accept a set of aligned sequences as input, which facilitates multiple sequence alignment by some iterative procedures. Dedicated to Professor Akiyoshi Wada on the occasion of his 60th birthday.     

Parallel evolution in radiation ofOhomopterus ground beetles inferred from mitochondrial ND5 gene sequences

Molecular phylogenetic analyses using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences representing all 15 species and the majority of subspecies or races of theOhomopterus ground beetles from all over the Japanese archipelago have uncovered a remarkable evolutionary history. Clustering of the species in the molecular phylogenetic tree is linked to their geographic distribution and does not correlate with morphological characters. Taxonomically the same species or the members belonging to the same species-group fall out in more than two different places on the ND5 tree. Evidence has been presented against a possible participation of ancestral polymorphism and random lineage sorting or of hybrid individuals for the observed distribution of mitochondrial DNA haplotypes. The most plausible explanation of our results is that parallel evolution took place in different lineages. Most notably,O. dehaanii, O. yaconinus, andO. japonicus in a lineage reveal almost identical morphology with those of the same species (or subspecies) but belonging to the phylogenetically remote lineages.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession numbers D50711-DD-50733 and D87131-D87186.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture