Physical identification of branched intron side-products of splicing in Trypanosoma brucei.

Every mRNA in trypanosomes consists of two exons, a common 5' capped mini-exon or spliced leader and a coding-exon. All evidence suggests that the exons are joined by trans-splicing of two individual precursor RNAs, the mini-exon donor RNA or spliced leader precursor RNA (medRNA) and the pre-mRNA. We studied intermediates of the splicing reaction using denaturing two-dimensional PAGE and structurally identified a group of small (approximately 180-300 nt) non-polyadenylated, Y-shaped branched RNAs. The branched Y-shaped RNAs contain the 105 nt medRNA derived intron, joined in a 2'-5' phosphodiester bond to small heterogeneously sized RNAs. These non-polyadenylated branched Y-shaped RNA molecules are analogous to the lariat shaped introns of higher eukaryotes and presumably represent the released intron-like by-products of a trans-splicing reaction which joins the mini-exon and the major coding-exon.     

Transposition of the Streptococcus lactis ssp. lactis Z270 lactose plasmid to pVA797: demonstration of an insertion sequence and its relationship to an inverted repeat sequence isolated by self-annealing

The lactose plasmid pUCL22 of the single plasmid strain Streptococcus lactis ssp. lactis Z270 was demonstrated to fuse with the heterologous conjugative plasmid pVA797. The fusion of pUCL22 with pVA797 occurred by recombination between a specific sequence of pUCL22 and different sites of pVA797. The cointegrates of pUCL22::pVA797 were unstable: in the absence of lactose selection, they segregated plasmids that corresponded to pVA797 enlarged by one sequence of 1.2 kb, common to all derivative plasmids. This resolution sequence (RS) was shown to originate in the 9.7 kb BstEII restriction fragment of pUCL22 and to duplicate during replicon fusion. In addition, after nuclease S1 treatment of pUCL22 DNA, a self-annealing sequence was isolated; the two copies of this inverted repeat (IR) sequence were located on the 18 kb BamHI segment of the plasmid. This latter sequence was distinct from the RS with which it hybridized weakly. The RS was responsible for the transposition of the entire lactose plasmid; the role of the IR remains to be elucidated.     

Effects of ATP on Phosphatidylinositol-Phospholipase C and Inositol 1-Phosphate Accumulation in Rat Brain Synaptosomes

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.     

Two specific topoisomerase II inhibitors prevent replication of human cytomegalovirus DNA: an implied role in replication of the viral genome.

In this study, we show that human cytomegalovirus DNA synthesis is inhibited in infected confluent human embryonic lung cells treated with the DNA-intercalative topoisomerase II inhibitor 4-9'-(acridinylamino)methanesulfon-m-anisidide (m-AMSA). Similar inhibitory effects were observed with VM-26, a nonintercalative topoisomerase II inhibitor. This antiviral effect is not attributable to cytotoxic effects per se. Furthermore, m-AMSA appears to have a notably irreversible inhibitory effect on human cytomegalovirus DNA replication. No inhibition of viral DNA synthesis was observed with o-AMSA, a DNA-intercalative isomer of m-AMSA that does not inhibit topoisomerase II.     

Detection of a single-copy gene on plant chromosomes by in situ hybridization

Summary DNA recombinant technology, combined with improvements in hybridization efficiency and quality of chromosome spreads, has made the method of in situ hybridization a reliable tool for gene mapping used by mammalian cytogeneticists to complement other methods. By appropriate alterations of the method, we demonstrate that detection of unique genes can be achieved along plant chromosomes despite some inherent disadvantages of the plant material. Using genomic subclones homologous to 6.6 kb of the single-copy chalcone synthase gene in parsley, we report the first example of chromosomal detection and localization of a unique endogenous gene in plants.     

Effect of Light on Sterol Changes in Medicago sativa

Most vascular plants contain Δ⁵-sterols as the predominant type; however, a few species such as Medicago sativa, have mainly Δ⁷-sterols. The Δ⁷-sterols of alfalfa are isomers of the common Δ⁵-sterols and are generally assumed to be their immediate precursors. Light had a significant influence on the sterol status of M. sativa. High light intensity and a long day favored the accumulation of dihydrospinasterol; a short day and low light intensity, particularly darkness, favored spinasterol accumulation. These data for Δ⁷-sterol plants agree with those reported for Δ⁵-sterol plants; light favors the accumulation of the monounsaturated 29 carbon sterols and darkness favors the accumulation of the diunsaturated sterols. Proposed is a mechanism to explain the effect of light on the accumulation of Δ⁷- and Δ⁵-sterols.     

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.