Bacteriophage P22 accessory recombination function.

The accessory recombination function (arf) gene of bacteriophage P22 is located immediately upstream of the essential recombination function (erf) gene. Three mutant alleles of arf were constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation. The deletion mutant phages are partially defective in homologous recombination and plaque formation in wild-type and recA hosts; their defects are more severe in recB and recA recB hosts. The amber mutant phage exhibits the same growth phenotypes in nonsuppressing hosts, but not in an amber-suppressor host. Plasmids that express arf complement the growth defect of arf- phages. These plasmids stimulate erf-mediated recombination; they were also found to cause a small stimulation of recA-recBCD-mediated homologous recombination of phage lambda.

Genetic structure of the bacteriophage P22 PL operon.

The sequence of 1416 base-pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combination from Plac UV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to lambda ral and cIII, respectively. P22 kil, like lambda kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and lambda kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream from erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the lambda red genes from plasmids. Sequences that include the stop codon for gene 17 may form a small stem-loop structure and are nearly identical to lambda sequences that contain the stop codon for ssb, which is near lambda tL 2b. Plasmids that include the P22 structure negatively regulate kil gene expression in cis.

Conformational changes in Newcastle disease virus fusion glycoprotein during intracellular transport.

The migration on polyacrylamide gels of nascent (pulse-labeled) and more processed (pulse-labeled and then chased) forms of nonreduced Newcastle disease virus fusion glycoprotein were compared. Results are presented which demonstrate that pulse-labeled fusion protein, which has an apparent molecular weight of 66,000 under reducing conditions (Collins et al., J. Virol. 28: 324-336), migrated with an apparent molecular weight of 57,000 under nonreducing conditions. This form of the Newcastle disease virus fusion protein has not been previously detected. This result suggests that the nascent fusion protein has extensive intramolecular disulfide bonds which, if intact, significantly alter the migration of the protein on gels. Furthermore, upon a nonradioactive chase, the migration of the fusion protein in polyacrylamide gels changed from the 57,000-molecular-weight species to the previously characterized nonreduced form of the fusion protein (molecular weight, 64,000). Evidence is presented that this change in migration on polyacrylamide gels is due to a conformational change in the molecule which is likely due to the disruption of some intramolecular disulfide bonds: Cleveland peptide analysis of the pulse-labeled nonreduced fusion protein (molecular weight, 57,000) yielded a pattern of polypeptides quite different from that obtained from the more processed form of the fusion protein (molecular weight, 64,000). However, the pattern of polypeptides obtained from the nonreduced 64,000-molecular-weight species was quite similar to that obtained from the fully reduced nascent protein (molecular weight, 66,000). This conformational change occurred before cleavage of the molecule. To determine the cell compartment in which the conformational change occurs, use was made of inhibitors which block glycoprotein migration at specific points. Monensin allowed the appearance of the 64,000-molecular-weight form of the fusion protein, whereas carboxyl cyanide m-chlorophenylhydrazine blocked the appearance of the 64,000-molecular-weight form of the fusion protein. Thus, the fusion protein undergoes a conformational change as it moves between the rough endoplasmic reticulum and the medial Golgi membranes.

Intracellular processing of the Newcastle disease virus fusion glycoprotein.

The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site (Nagai et al., Virology 69:523-538, 1976) into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with [3H] fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.

Influence of ethanol on human T-lymphocyte migration.

Because ethanol consumption is associated with increased susceptibility to infection, we examined the effects of ethanol and its metabolite acetaldehyde on human T-lymphocyte migration, an important functional component of cellular inflammatory responses. With a modified Boyden chamber system, ethanol at 0.25% and 0.50% (vol/vol) inhibited spontaneous motility of human T-lymphocytes, in a noncytotoxic manner, to 65% +/- 7% (mean +/- SEM) and 62% +/- 7% of control values of migration, respectively. When T-lymphocyte migration was stimulated by colchicine (10(-5) mol/L), incubation with ethanol (0.25% and 0.50%, vol/vol) decreased migration to 80% +/- 4% and 66% +/- 8% of control values, respectively. Similar degrees of inhibition of migration were obtained with acetaldehyde at concentrations five to 10 times less than ethanol. Ethanol was similarly capable of inhibiting T cell migration induced by dibutyryl cyclic guanosine monophosphate, but it had no effect on stimulated migration induced by a human chemokinetic lymphokine. Our study demonstrates that ethanol, at concentrations achievable in vivo, is capable of depressing T-lymphocyte migration. This effect might contribute to the immunosuppression associated with ethanol consumption.