B subunit of E. coli enterotoxin as adjuvant and carrier in oral and skin vaccination.

Mucosal sites are one of the main natural ports of entry into the body. Stimulation of a local response by antibodies as the systemic protection may enhance the efficacy of non-living vaccines, and allow for vaccination by subunit vaccines without the need for injection. Mucosal or skin vaccination necessitates a suitable adjuvant and carrier. Escherichia coli heat-labile enterotoxin (LT) and its B subunit (LTB) have been found to be effective adjuvants. The aim of this study was to efficiently produce and purify recombinant LTB (brLTB), and examine its adjuvant and carrier properties. The gene encoding LTB was cloned and expressed in E. coli, and the product was found to have a pentameric form with the ability to bind the cell receptor, GM1 ganglioside. A one-step method for efficient purification and concentration of brLTB was developed. Both oral and intramuscular vaccination with purified brLTB yielded high antibody titers, which detected the whole toxin. In an attempt to test its adjuvant characteristics, brLTB was mixed with either BSA or a recombinant protein (rKnob of egg drop syndrome adenovirus) and delivered intramuscularly, orally or transcutaneously. The addition of brLTB significantly elevated the antibody response in groups vaccinated orally and transcutaneously, but had no influence in injected groups. Vaccination with another recombinant protein, (viral protein 2 of infectious bursal disease virus) supplemented with brLTB did not elevate the antibody response, as compared to vaccination with the antigen alone. These results demonstrate that the addition of brLTB makes oral and transcutaneous vaccination with protein antigens possible.

A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance.

Lithium acetate transformation and electroporation were applied to the biocontrol yeast, Candida oleophila. The hygromycin B resistance gene, flanked by the phosphoglycerate kinase promoter and terminator of Candida tropicalis, served as the genetic selection marker. The transformation efficiency of electroporation was almost 400 times more efficient than that of the lithium acetate method. While incorporation of DNA, flanked by a sequence endogenous to C. oleophila, transpired apparently by homologous recombination, the integration of DNA (that did not contain C. oleophila DNA) occurred at random. Whereas transformants were observed with a linear segment of the plasmid, none were detected with the undigested plasmid. This system provides both a tool for the molecular analysis of the biocontrol mechanism of C. oleophila and a means of tagging C. oleophila for field studies.

Transfer of antibodies elicited by baculovirus-derived VP2 of a very virulent bursal disease virus strain to progeny of commercial breeder chickens.

A baculovirus-derived recombinant VP2 (rVP2) subunit vaccine elicited anti-infectious bursal disease virus (IBDV) antibodies in commercial flocks. The induced antibody levels were similar to those evoked against IBDV by a commercial vaccine. The levels remained higher than that of the negative controls for at least four and a half months in commercial chickens. The antibodies were also transferred to their offspring and were detected in the blood of the progeny for at least 20 days after hatching. These results, along with former data, that show that antibodies elicited by baculovirus rVP2 confer protection to chickens from IBDV [J. Pitcovski et al. (1996), Insect cell-derived VP2 of infectious bursal disease confers protection against the disease in chickens. Avian Diseases, 40, 753-761], imply that the baculovirus-derived rVP2 subunit may serve as a successful vaccine for commercial breeding flocks.

Viral protein 1 sequence analysis of three infectious bursal disease virus strains: a very virulent virus, its attenuated form, and an attenuated vaccine.

Comparisons between sequences of very virulent, virulent, and attenuated strains of the infectious bursal disease virus (IBDV) may indicate sites on the genome co-inciding with virulence. In an attempt to detect if such sites exist on the coding region of segment B, viral protein 1 (VP1) (encoded for by segment B) of a very virulent Israeli virus, IL3; its attenuated strain, IL4; and the attenuated Winterfield vaccine 2512 were cloned and sequenced. A comparison was made among them and with six other published sequences of segment B. Six nucleic acids distinguished between IL3 and IL4, three of which were predicted to be expressed as amino acids. A striking similarity between the VP1 sequences of 2512 and P2 (an attenuated German strain) was discovered. Although conclusions could not be drawn concerning attenuation sites on VP1, the analysis performed on the VP1 sequences of the two Israeli strains and the Winterfield 2512 strain sheds light on the phylogeny of IBDV and contributes to the accumulating information that may lead to the identification of virulence-related sites of this virus.

Multidrug resistance in Candida albicans: disruption of the BENr gene.

The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.

Meiotic nondisjunction and recombination of chromosome III and homologous fragments in Saccharomyces cerevisiae.

A yeast strain, in which nondisjunction of chromosome III at the first meiotic division could be assayed, was constructed. Using chromosome fragmentation plasmids, chromosomal fragments (CFs) were derived in isogenic strains from six sites along chromosome III and one site on chromosome VII. Whereas the presence of the CFs derived from chromosome III increased considerably the meiosis I nondisjunction of that chromosome, the CF derived from chromosome VII had no effect on chromosome III segregation. The effects of the chromosome III-derived fragments were not linearly related to fragment length. Two regions, one of 12 kb in size located at the left end of the chromosome, and the other of 5 kb, located at the center of the right arm, were found to have profound effects on chromosome III nondisjunction. Most disomics arising from meioses in strains containing chromosome III CFs did not contain the CF; thus it appears that the two chromosome III homologs had segregated away from the CF. Among the disomics, recombination between the homologous chromosomes III was lower than expected from the genetic distance, while recombination between one of the chromosomes III and the fragment was frequent. We suggest that there are sites along the chromosome that are more involved than others in the pairing of homologous chromosomes and that the pairing between fragment and homologs involves recombination among these latter elements.

A short chromosomal region with major roles in yeast chromosome III meiotic disjunction, recombination and double strand breaks.

A multicopy plasmid was isolated from a yeast genomic library, whose presence resulted in a twofold increase in meiotic nondisjunction of chromosome III. The plasmid contains a 7.5-kb insert from the middle of the right arm of chromosome III, including the gene THR4. Using chromosomal fragments derived from chromosome III, we determined that the cloned region caused a significant, specific, cis-acting increase in chromosome III nondisjunction in the first meiotic division. The plasmid containing this segment exhibited high spontaneous meiotic integration into chromosome III (in 2.4% of the normal meiotic divisions) and a sixfold increase (15.5%) in integration in nondisjunctant meioses. Genetic analysis of the cloned region revealed that it contains a "hot spot" for meiotic recombination. In DNA of rad50S mutant cells, a strong meiosis-induced double strand break (DSB) signal was detected in this region. We discuss the possible relationships between meiosis-induced DSBs, recombination and chromosome disjunction, and propose that recombinational hot spots may be "pairing sites" for homologous chromosomes in meiosis.

Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae.

We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S. cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known 'hot spots' for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.