Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase.

The cellular 300 kDa protein known as p300 is a target for the adenoviral E1A oncoprotein and it is thought to participate in prevention of the G0/G1 transition during the cell cycle, in activation of certain enhancers and in the stimulation of differentiation pathways. In order to determine the exact function of p300, as a first step we constructed a simple assay system for the selection of a potential target site of a hammerhead ribozyme in vivo. For the detection of ribozyme-mediated cleavage, we used a fusion gene (p300-luc) that consisted of the sequence encoding the N-terminal region of p300 and the gene for luciferase, as the reporter gene. We were also interested in the correlation of the GUX rule, for the triplet adjacent to the cleavage site, with ribozyme activity in vivo. Therefore, we selected five target sites that all included GUX The rank order of activities in vitro indeed followed the GUX rule; with respect to the kcat, a C residue as the third base (X) was the best, next came an A residue and a U residue was the worst (GUC > GUA > GUU). However, in vivo the tRNA(Val) promoter-driven ribozyme, targeted to a GUA located upstream of the initiation codon, had the highest inhibitory effect (96%) in HeLa S3 cells when the molar ratio of the DNA template for the target p300 RNA to that for the ribozyme was 1:4. Since the rank order of activities in vivo did not conform to the GUX rule, it is unlikely that the rate limiting step for cleavage of the p300-luc mRNA was the chemical step. This kind of ribozyme expression system should be extremely useful for elucidation of the function of p300 in vivo.     

Purification and characterization of microbial gellan lyase.

Gellan lyase was purified from the culture fluid of soil samples incubated in a medium containing gellan as a sole carbon source. The enzyme was a monomer with a molecular mass of 140 kDa and was most active at pH 7.5 and 45 degrees C. The enzyme was highly specific to gellan and lowered the viscosity of the polymer.     

Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization

Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully applied to the specimens, revealing one ribosomal RNA gene cluster, or nucleolus organizer region (NOR) in the genome of each fungus. A long attenuated chromatid thread expanding from a condensed metaphase chromosome, which had been called a thread-like structure in B. cinerea, was proved to be an NOR. This is the first report of the successful application of FISH to the chromosomes of filamentous fungi.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Photosynthetic oxygen evolution is stabilized by cytochrome c550 against heat inactivation in Synechococcus sp. PCC 7002.

We investigated the factors responsible for the heat stability of photosynthetic oxygen evolution by examining thylakoid membranes from the cyanobacterium Synechococcus sp. PCC 7002. We found that treatment of the thylakoid membranes with 0.1% Triton X-100 resulted in a remarkable decrease in the heat stability of oxygen evolution, and that the heat stability could be restored by reconstituting the membranes with the components that had been extracted by Triton X-100. The protein responsible for the restoration of heat stability was purified from the Triton X-100 extract by two successive steps of chromatography. The purified protein had a molecular mass of 16 kD and exhibited the spectrophotometric properties of a c-type Cyt with a low redox potential. The dithionite-minus-ascorbate difference spectrum revealed an alpha band maximum at 551 nm. We were able to clone and sequence the gene encoding this Cyt from Synechococcus sp. PCC 7002, based on the partial amino-terminal amino acid sequence. The deduced amino acid sequence revealed a gene product consisting of a 34-residue transit peptide and a mature protein of 136 residues. The mature protein is homologous to Cyt c550, a Cyt with a low redox potential. Thus, our results indicate that Cyt c550 greatly affects the heat stability of oxygen evolution.     

Phytochrome-Mediated Photoorientation of Chloroplasts in Protonemal Cells of the Fern Adiantum Can be Induced by Brief Irradiation with Red Light

Measuring the ratio of the number of photooriented chloroplaststo the total number of chloroplasts, we found that photoorientationof chloroplasts in protonemata of the fern Adiantum capillus-veneriscould be induced by brief irradiation with polarized red light.After irradiation with red light (R) of 3 or 10 min, orientationalmovement was detected as early as 10 min after the irradiation;it continued during the subsequent dark period for 30–60min, after which chloroplasts gradually dispersed again. WhenR-treated protonemata were irradiated briefly with a second10-min pulse of R, 60 min after the onset of the first irradiation,the orientational response of chloroplasts was again observed.Typical red/far-red photoreversibility was apparent in the response,indicating the involvement of phytochrome. By contrast, irradiationwith polarized blue light for 10 min was ineffective, whileirradiation with blue light (B) at the same fluence for a longerperiod of time clearly induced the photoorientation of chloroplasts.It is likely that longterm irradiation is necessary for theresponse mediated by a blue-light receptor. When protonemata were irradiated with far-red light (FR) immediatelyafter R or after a subsequent dark period of 10 min, the magnitudeof the orientational response was smaller and chloroplasts dispersedmore quickly than those exposed to R alone. When FR was appliedat 50 min, when the response to R had reached the maximum level,chloroplasts again dispersed rapidly to their dark positions.These results indicate that P_FR not only induces the photoorientationmovement of chloroplasts but also fixes the chloroplasts atthe sites to which they have moved as a result of photoorientation. (Received June 2, 1993; Accepted January 11, 1994)     

Photosynthetic Adaptation to High Temperature Associated with Thylakoid Membranes of Synechococcus PCC7002

Photosynthetic adaptation to high temperature was investigatedin intact cells and isolated thylakoid membranes of the cyanobacterium,Synechococcus PCC7002. In intact cells, the thermal stabilityof photosynthesis and photosystem 2-mediated electron transportfrom H₂O to 1,4-benzoquinone changed in concert with growthtemperature. The photosystem 2-mediated electron transport fromH₂O to phenyl-1,4-benzoquinone showed greater thermal stabilityin thylakoid membranes isolated from cells which had adaptedto high temperature than in those from non-adapted cells. Enhancedthermal stability was also observed in the thylakoid membranesin the transport of electrons from H₂O to 2,6-dichlorophenolindophenolbut not in the transport of electrons from diphenylcarbazideto 2,6-dichlorophenolindophenol. These observations suggestthat oxygen-evolving sites acquire enhanced thermal stability,and that factors which are responsible for thermal stabilityremain in isolated thylakoid membranes. (Received October 30, 1992; Accepted December 18, 1992)     

Functional analysis of the γ-glutamylcysteine synthetase of Escherichia coli B: effect of substitution of His-150 to Ala

A high expression system of the -glutamylcysteine synthetase gene (gshl) of Escherichia coli B was constructed, and rapid purification of GSH-I was performed. The active site of GSH-I was analysed by chemical modification, and Lys, Arg and His residues seemed to be involved in the active site of the enzyme. Among them, His residues were substituted to Ala by site-directed mutagenesis, and His-150 was found to be essential for the activity of GSH-I. Correspondence to: A. Kimura     

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.