The Drosophila double-timeS mutation delays the nuclear accumulation of period protein and affects the feedback regulation of period mRNA.

The Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein. Here, the circadian phenotype of a short-period dbt mutant allele (dbt(S)) was examined. The circadian period of the dbt(S) locomotor activity rhythm varied little when tested at constant temperatures ranging from 20 to 29 degrees C. However, per(L);dbt(S) flies exhibited a lack of temperature compensation like that of the long-period mutant (per(L)) flies. Light-pulse phase-response curves were obtained for wild-type, the short-period (per(S)), and dbt(S) genotypes. For the per(S) and dbt(S) genotypes, phase changes were larger than those for wild-type flies, the transition period from delays to advances was shorter, and the light-insensitive period was shorter. Immunohistochemical analysis of per protein levels demonstrated that per protein accumulates in photoreceptor nuclei later in dbt(S) than in wild-type and per(S) flies, and that it declines to lower levels in nuclei of dbt(S) flies than in nuclei of wild-type flies. Immunoblot analysis of per protein levels demonstrated that total per protein accumulation in dbt(S) heads is neither delayed nor reduced, whereas RNase protection analysis demonstrated that per mRNA accumulates later and declines sooner in dbt(S) heads than in wild-type heads. These results suggest that dbt can regulate the feedback of per protein on its mRNA by delaying the time at which it is translocated to nuclei and altering the level of nuclear PER during the declining phase of the cycle.

Molecular aspects of complement-mediated bacterial killing. Periplasmic conversion of C9 from a protoxin to a toxin.

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.

Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.

Association of terminal complement proteins in solution and modulation by suramin.

The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering. Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of -8.3 kcal/mol and a dissociation constant Kd of 0.6 microM (at 20 degrees C) that was ionic strength- and temperature-dependent. A van't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees = -12.9 kcal/mol and DeltaS degrees = -15.9 cal mol-1 deg-1, suggesting that electrostatic forces play a prominent role in the interaction of C8 with C9. Native C8 also formed a heterodimer with C5, and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction. Suramin induced high-affinity trimerization of C8 (Kd = 0.10 microM at 20 degrees C) and dimerization of C9 (Kd = 0.86 microM at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to prevent complement-mediated hemolysis. Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers. Oligomerization could be reversed by high suramin concentrations, but 8-aminonaphthalene-1,3,6- trisulfonate (ANTS2- ), which mimics half a suramin molecule, could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins.

The expression of hemolytically active human complement protein C9 in mammalian, insect, and yeast cells.

The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.