Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product.

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.     

A binocular model for the simple cell

We authors propose a mathematical model for simple cell binocular response. It comprises two Gabor-type receptive fields (RF) having the same RF center, preferred spatial frequency, and preferred orientation. The model integrates the equally weighted signals from both eyes and performs a threshold operation. Poggio and Fischer (1977) classified binocular disparity cells in the striate cortex into four groups: tuned excitatory (TE), tuned inhibitory (TI), near, and far cells. They also found that most of the TE cells are ocularly balanced and that the other three types are usually unbalanced. This model can imitate these four types of disparity sensitivities and their ocular dominance tendency. We perform model fittings to Poggio's data using the “simulated annealing” method and discuss parameter dependence of the model's response. The model can also respond with exceptional disparity sensitivity: i.e., flat type, alternating type, and intermediate type.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

Effects of Physical Parameters upon Protoplast Electrofusion

The effects of three physical parameters upon protoplast electrofusionwere studied using protoplasts from cultured cells of Coptisjaponica and Euphorbia millii. The osmotic potential of themedium did not appreciably affect the AC-field-induced protoplast-pairformation, but significantly influenced the fusion process ofthe paired protoplasts in response to DC pulses. The optimumosmotic potential was 0.55 to 0.60 Osm/kg H₂O in our system.The density of the medium markedly influenced both pair formationand fusion process. The optimum density was 1.13 to 1.14 g/cm₃,and at this density the yield of the fused protoplasts increasedto more than twice that of the control (1.10 g/cm₃). Hydrophiliccoating of the bottom surface of the chamber with Gellan gumor polyacrylamide gel was also effective for both pair formationand the fusion process, while coating with hydrophobic siliconewas entirely inhibitory. Possible interpretations of the effectsof these physical parameters upon protoplast electrofusion arepresented. ¹Permanent address: Biochemical Research Laboratories, KanegafuchiChemical Industry Co., Ltd., Takasago, Hyogo 676, Japan. (Received December 21, 1987; Accepted March 18, 1988)     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.