Alternative splicing produces a constitutively active form of human SREBP-1

We identified a novel alternative splicing event that constitutively produces a truncated active form of human sterol regulatory element-binding protein 1 (SREBP-1). A cDNA of this splicing variant (named SREBP-1Δ) contains a translational stop codon-encoding exon sequence between exons 7 and 8. It produces SREBP-1aΔ (470 a.a.) and SREBP-1cΔ (446 a.a.) proteins that lack transmembrane and C-terminal regulatory sequences necessary for localization of SREBP-1 to the endoplasmic reticulum. A luciferase reporter assay showed that SREBP-1aΔ and SREBP-1cΔ transactivated lipogenic gene promoters to the same extent as that induced by N-terminal active fragments of SREBP-1a and SREBP-1c, respectively. SREBP-1Δ mRNA is expressed in human cell lines as well as adipose and liver tissues. Expression levels ranged from 5% to 16% of total SREBP-1 expression. The ratio of SREBP-1Δ expression to total SREBP-1 expression in HepG2 cells was not affected by either insulin or high glucose treatment.     

RNA extraction from various recalcitrant plant tissues with a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment

High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction.     

Reaction System of Haemophilus,Superhelical DNA-relaxing Enzyme is Useful for Screening of DNA-binding Substance

Several substances with antioxidant activity were isolated from the mixture of dehydroascorbic acid and tryptophan when reacted together in ethanol. One of the main antioxidant products was obtained in crystalline form from an n-butanol extract of the reaction mixture by means of Sephadex column chromatography followed by HPLC with a reversed phase column. ¹H- and ¹³C-NMR of the product and its acetate showed its structure as a condensate of dehydroascorbic acid and tryptophan with each single molecule involving a C-spiro structure. By a POV test the activity of this substance was about two-thirds of that of BHA on a molar basis, and the activity of the reaction mixture is greatly attributable to this substance.     

Effects of Panicle Removal on the Photosynthetic Characteristics of the Flag Leaf of Rice Plants during the Ripening Stage

All panicles were removed from rice plants (Oryza sativa L.)at anthesis and the effects of this treatment on photosyntheticgas-exchange rates and the underlying biochemical propertiesof the blade of the flag leaf were examined during senescence.Panicle removal retarded the decrease in photosynthesis in theflag leaf during senescence. Similarly, the levels of ribulose-l,5-bisphosphatecarboxylase (Rubisco), chlorophyll and Cyt f, and the activitiesof sucrose-phosphate synthase (SPS) and ADP-glucose pyrophosphorylasein panicle-free plants remained relatively high during leafsenescence and decreased more slowly than those in the controlplants. Regression analysis showed no difference between thetreated and untreated plants in the relationship between CO₂-limitedphotosynthesis (measured at an intercellular CO₂ pressure of15 Pa) and Rubisco content, or in the relationships betweenCO₂-saturated photosynthesis (measured at an intercellular CO₂pressure above 60 Pa), Cytf content and SPS activity. Theseresults indicated that the change in photosynthesis caused bypanicle removal at anthesis could be explained by the changesin the amounts and/or activities of the major biochemical participantsin photosynthesis. The temporary accumulation of starch and sucrose in the flagleaf was observed in the panicle-free plants, but the accumulationwas not significant. However, in the panicle-free plants, theweight of shoots, excluding panicles, increased by 200% andthat of roots increased by 150% at the harvesting stage. Thus,it appears that, since the effective translocation of photosynthateto other organs, such as late tillers and roots, can occur,the removal of panicles has no effect on the relationship betweenthe rate of photosynthesis and the levels of the major biochemicalparticipants in photosynthesis. (Received January 7, 1995; Accepted March 15, 1995)     

New Suppressors of Frameshift Mutations in SALMONELLA TYPHIMURIUM

Several new types of suppressor mutants have been isolated. These were identified among revertants of mutants originally generated by mutagens other than the acridine-derived ICR191. The new suppressors correct mutations other than those with runs of C or G which are recognized by the previously described suppressors. Several frameshift mutations are corrected by more than one suppressor type. Apparently, the DNA base sequence near these mutant sites includes sites of action for several distinct suppressor types.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.     

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).     

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S₁ nuclease cleavage. The S₁-cleavage occurred once at these sites. In the absence of added Mg²⁺, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S₁, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S₁-cleavable, unbasepaired sites.     

Degradation of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase in the Lysates of the Chloroplasts Isolated Mechanically from Wheat (Triticum aestivum L.) Leaves

Intact chloroplasts were isolated mechanically from the primaryleaves of 8- to 12-day old seedlings of wheat (Triticum aestivumL.) and purified by Percoll gradient centrifugation. The chloroplastswere lyzed by osmotic shock and the reaction mixtures containingthe lysates were incubated in the pH range of 5.3 to 9.4 at37°C. The degradation of ribulose-l,5-bisphosphate carboxylase/oxygenase(RuBisCO, EC 4.1.1.39 [EC] ) and its degradation products in the mixtureswere examined by using SDS-polyacrylamide gel electrophoresis.RuBisCO-hydrolase activity in the lysates was very weak, andit was difficult to assess the activity by measuring the lossof the amount of the large subunit of RuBisCO on the gels afterstaining with Coomassie Brilliant Blue. By using immunoblottingmethod, however, degradation products of RuBisCO could be detectedin the reaction mixtures. The hydrolase activity was pronouncedin the presence of 0.1 % (w/v) of SDS in the reaction mixtures.Among the products, the 35 kDa fragment was conspicuous andfound in the wide range of pHs. This degradation of RuBisCOwas inhibited in the presence of leupeptin and N-ethylmaleimide. (Received October 3, 1988; Accepted November 25, 1988)