By using a gene-specific fragment from the hemolytic phospholipase C (PLC) gene of Pseudomonas aeruginosa as a probe and data from Southern hybridizations under reduced stringency conditions, we cloned a 4.2-kb restriction fragment from a beta-hemolytic Pseudomonas cepacia strain which expressed hemolytic and PLC activities in Escherichia coli under the control of the lac promoter. It was found, by using a T7 phage promoter-directed expression system, that this DNA fragment carries at least two genes. One gene which shares significant DNA homology with both PLC genes from P. aeruginosa encodes a 72-kDa protein, while the other gene encodes a 22-kDa protein. When both genes on the 4.2-kb fragment were expressed from the T7 promoter in the same cell, hemolytic and PLC activities could be detected in the cell lysate. In contrast, when each individual gene was expressed in different cells or when lysates containing the translated products of each separate gene were mixed, neither hemolytic activity nor PLC activity could be detected. Clinical and environmental isolates of P. cepacia were examined for beta-hemolytic activity, PLC activity, sphingomyelinase activity, and reactivity in Southern hybridizations with a probe from P. cepacia which is specific for the larger gene which encodes the 72-kDa protein. There were considerable differences in the ability of the different strains to express hemolytic and PLC activities, and the results of Southern DNA-DNA hybridizations of the genomic DNAs of these strains revealed considerable differences in the probe-reactive fragments between highand medium-stringency conditions as well as remarkable variation in size and number of probe-reactive fragments among different strains. Analysis of the genomic DNAs from hemolytic and nonhemolytic variants of an individual strain (PC-69) by agarose gel electrophoresis, Southern hybridization, and transverse alternating pulsed field gel electrophoresis suggests that the conversion of the hemolytic phenotype to the nonhemolytic phenotype is associated with either the loss of a large plasmid (>200 kb) or a large deletion of the chromosome of P. cepacia PC-69.

To understand mechanisms involved in sex-specific gene expression in Schistosoma mansoni, a cDNA (fs800) was isolated that hybridized to an 800 nucleotide mRNA present in high levels only in mature female worms. The fs800 cDNA sequence was characterized by two long open reading frames and central stretches of repeated amino acids. Fs800 did not share similarities with other known sequences in computer searches. In situ hybridization, however, revealed that the mRNA corresponding to fs800 was found only in female vitelline cells, suggesting that the product of this gene may be involved in the production or function of eggs. Fs800 is developmentally regulated as expression of this gene is dependent on the maturity of female worms. Furthermore, during in vitro culture, when female worms are known to stop egg production, expression of fs800 selectively ceased.

We typed 40 isolates of Pseudomonas cepacia obtained from patients involved in a single outbreak using pulsed field gel electrophoresis and ribotyping. All isolates from the majority of the patients, 16 of 18 (89%), were included in a single group. These typing methods should aid in the clarification of the epidemiology of infection with P. cepacia.

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The combined stimulus of FITCI-conjugated autologous cells plus either allogeneic cells, soluble antigens, or T cell-derived helper factor(s) triggers the in vitro differentiation of FITCI altered-self-reactive human CTL. Fractionation of responder cells into highly purified lymphocyte subpopulations and complement-mediated lysis of FITCI altered-self-reactive killer cells with rabbit anti-human T cell antibodies demonstrated that both the precursor and effector cell responsible for FITCI altered-self-cytolysis are T lymphocytes. Killer cell specificity was investigated in three experimental protocols: 1) the CTL generated were found to efficiently lyse FITCI but not TNP-modified autologous targets, whereas in reciprocal experiments, TNP altered-self-reactive CTL lyse TNP but not FITCI-derivitized autologous cells; 2) T cell-mediated lysis of 51Cr-labeled FITCI-derivitized autologous targets was specifically inhibited by nonradiolabeled FITCI but not TNP-modified autologous cells; and 3) FITCI altered self-reactive CTL specifically lyse some but not other FITCI-modified allogeneic targets. Taken together, these results show that although the FITCI altered-self-reactive human CTL generated are hapten specific, they appear to recognize more than hapten alone on the target cell surface. Moreover, the observation that soluble antigens, allogeneic cells, and T cell helper factor(s) can facilitate hapten-specific CTL responses to both TNP and FITCI-conjugated autologous cells suggests that these helper stimuli act by preferentially triggering the differentiation of CTL precursors that have specifically interacted with chemically modified autologous stimulators.