Similar-sized daughter-strand gaps are produced in the leading and lagging strands of DNA in UV-irradiated E. coli uvrA cells.

The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps. The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes. The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes. After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel. It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb. No gaps larger than 0.5 kb were detected.     

1,10-Phenanthroline-copper, a footprinting reagent for single-stranded regions of RNAs.

The 1,10-phenanthroline-cuprous complex (OP-Cu) with hydrogen peroxide as a coreactant nicks the single-stranded loops and bulges of RNA stem-loop structures more rapidly than the double-stranded stems. This chemical nuclease is therefore a useful footprinting reagent for these regions and can be used to monitor both intramolecular and intermolecular hybridization of single-stranded domains. The formation of A-form structures characteristic of either RNA-RNA or RNA-DNA duplexes inhibits scission because it blocks the binding site of the coordination complex in single-stranded loops and not because the oxidatively sensitive hydrogens of the ribose moiety are blocked. The C-4' and C-1' hydrogens are accessible to solvent in A-structures.     

Enhanced photorelaxation in aorta, pulmonary artery and corpus cavernosum produced by BAY K 8644 or N-nitro-L-arginine.

Segments of endothelium-denuded aorta, pulmonary arterial rings and strips of corpus cavernosum from rabbits were superfused with Krebs medium. Photorelaxation elicited by ultraviolet light (366 nm) was significantly enhanced by either BAY K 8644 (20 nM) or N-nitro-L-arginine (100 and 500 microM) and was associated with increased cyclic GMP. This action of both drugs was greater in pulmonary artery than aorta and corpus cavernosum and persisted in vascular rings for 90 min after drug removal. The effect was significantly attenuated by hemoglobin (10 microM) but was unaltered by superoxide dismutase (30 u/ml). The mechanism of such photosensitization is presently unclear.     

Kinetic evidence of microscopic states in protein folding.

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3<-->D2<-->D1<-->N0, in which N0 is the native state and D's are the three substates of the denatured form [Chen, H.M., You, J.L., Markin, V.S., & Tsong, T.Y. (1990) J. Mol. Biol. 220, 771-778; Chen, H.M., Markin, V.S., & Tsong, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the single- and the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25 degrees C, log tau 1(-1) (for the D1<-->N0 step) and log tau 2(-1) (for the D2<-->D1 step) vs pH show a square root of-shaped dependence on the final pH, with minimal values (tau 1(-1) of 0.56 s-1 and tau 2(-1) of around pH 3.9. The third relaxation tau 3 (for the D3<-->D2 step, 35 s) was independent of pH in the range 3.4-8.5. The square root of-shaped dependence on pH of log tau 1(-1) and log tau 2(-1) cannot be reproduced by the above but can be accounted for if each of N0, D1, and D2 is composed of many microscopic states in rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of MyoD1 activity by 5-aza-2'-deoxycytidine.

A mouse myogenic determination gene, MyoD1, was transfected into the human osteogenic sarcoma cell line TE85. Several stably transfected clones were isolated which, at low frequencies, formed multinucleated cells with the appearance of skeletal myotubes. Southern blot analysis confirmed the integration of multiple copies of the mouse MyoD1 gene, and Northern analysis and immunofluorescence confirmed its expression in the transfectants. Characterization of the transfectants showed that they expressed immunologically detectable myosin, desmin, mRNA for myogenin, and the delta subunit of the acetylcholine receptor. The cells assembled a functional contractile apparatus since they contracted in response to acetylcholine added to the culture medium. The presence of MyoD1 protein did not abrogate the expression of two genes active in bone cells but not in muscle cells. The transfected cells therefore displayed a chimeric phenotype by expressing simultaneously bone and muscle genes. Interestingly, treatment of the MyoD1 transfected cells with 5-aza-2'-deoxycytidine resulted in a substantial increase in the frequency of myogenic conversion. Thus, the methylation inhibitor increased the ability of MyoD1 to function as a trans-acting factor and activate the muscle phenotype.     

A new species ofAphidius [Hymenoptera] attacking the pea aphid,Acyrthosiphon pisum

Aphidius staryi Chen & Luhman n. sp. is described. The species was collected and introduced into California from Israel and Turkey byD. González. The new species is morphologically most similar toAphidius smithi, and keys toEady's urticae group.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.     

Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes.

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.