Large-scale analysis of the yeast genome by transposon tagging and gene disruption.

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

Immune responses to third stage larvae of Onchocerca volvulus in interferon-gamma and interleukin-4 knockout mice.

To shed clarity on the dichotomy of reported results relative to the significance of T helper-1 vs T helper-2 immune responses in onchocerciasis, we compared the survivability of Onchocerca volvulus third-stage larvae (L3) in immunized mice that had either a targeted disruption of the Interleukin-4 or Interferon-gamma gene. Treatment groups consisted of control mice and mice immunized with irradiated O. volvulus L3. All mice were challenged with diffusion chambers containing viable L3. Vaccinated IL-4-/- were unable to kill this larval target. In contrast, vaccinated INF-gamma-/- and C57BL/6 mice, exhibited high levels of killing, had elevated levels of IL-4 and significantly greater numbers of eosinophils in their diffusion chambers than the IL-4-/-. Whereas, levels of IFN-gamma in all three groups of immunized mice were equivalent to those of control mice, levels of IL-5 were elevated, even in the IL-4-/-, indicating that cytokines other than IL-4 were involved in its production. The protective immune response to third-stage larvae of O. volvulus in mice vaccinated with irradiated larvae has an absolute IL-4 requirement.

Maternal influence on susceptibility of offspring to Brugia malayi infection in a murine model of filariasis.

We have used the severe combined immunodeficient C.B-17-scid/scid mouse to investigate the influences of maternal immune status and parasite burden on the susceptibility (or resistance) of offspring to infection with the human filarial parasite, Brugia malayi. C.B-17-scid/scid mice are permissive for infection while immunocompetent C.B-17(-)+/+ mice are uniformly resistant. Reciprocal matings of C.B-17-scid/scid and C.B-17(-)+/+ mice were performed. The C.B-17-scid/scid females were either naive or infected with Brugia malayi. The resulting immunocompetent C.B-17-scid/+ and C.B-17(-)+/scid progeny were challenged at weaning with an intraperitoneal injection of Brugia malayi third stage larvae known to produce patent infection in > 95% of C.B-17-scid/scid mice. We observed that 40.0%l (34/85) of the immunocompetent offspring of C.B-17-scid/scid females x C.B-17(-)+/+ males were permissive for the growth and development of Brugia malayi larvae to adults. No difference was observed in susceptibility to infection between the progeny of infected or uninfected C.B-17-scid/scid mothers mated with C.B-17(-)+/+ fathers, arguing against acquired immunological tolerance to the parasite in the former. In marked contrast, only 4.8% (2/42) of the heterozygous progeny of wild type C.B-17(-)+/+ females mated with C.B-17-scid/scid males were permissive. These observations document conversion of a 'resistant' phenotype to a 'susceptible' phenotype by manipulation of maternal immune status and provide clear evidence of maternal influence on offspring susceptibility to infection with Brugia malayi.

Infectivity and normal development of third stage Brugia malayi maintained in vitro.

Shipment of infective-stage filarial larvae (L3s) usually has been accomplished by transporting living infected vectors or L3s cryopreserved in liquid nitrogen. Our objective was to find culture conditions for transporting L3s that would promote survival of Brugia malayi larvae without altering their capacity to infect susceptible animals. In preliminary studies we observed that Ham's nutrient mixture F-12, with antibiotics and 1% fetal calf serum, could support L3s without apparent development for at least 10 days. In order to evaluate the effect of culture temperatures on infectivity, fresh L3s were divided into groups that were either immediately injected into jirds (infectivity control) or incubated for 24, 48, or 120 hr in tightly sealed tubes maintained horizontally at either 0 C, 20 C, or 37 C, before they were injected into jirds. Necropsies were performed on the jirds 120-130 days after injection to recover and count adult worms. Levels of microfilaremia were also determined. We found that L3s held overnight at 0 C, although apparently viable, were unable to survive in jirds. However, larvae kept at 20 C and 37 C produced patent infections with adult worms in normal locations even after 120 hr of in vitro cultivation. There was no statistical difference in mean worm recovery or size of worms from jirds infected with freshly harvested L3s and jirds injected with larvae that were maintained overnight at 20 C or 37 C. When cultured L3s were shipped from Michigan to Connecticut by overnight air courier, along with infected living mosquitos, the L3s appeared to be 99% viable upon arrival. L3s shipped in F-12 produced patent infections in C.B.-17 scid/scid mice with worm recoveries comparable to those observed in mice injected with L3s freshly obtained from shipped mosquitos.

CD4+ T-lymphocytes are not required for murine resistance to the human filarial parasite, Brugia malayi.

Immunocompetent mice are nonpermissive for the development and maturation of the human filarial parasite, Brugia malayi. We and others have shown that the absence of T-lymphocytes, alone or in combination with B-lymphocytes, renders mice permissive to infection. In a previous study, we showed that mice lacking CD8+ T-lymphocytes are also completely nonpermissive for B. malayi, indicating that CD8+ T-lymphocytes are not an obligate requirement for resistance. In the present study, we have examined the role of CD4+ T-lymphocytes in resistance to filarial infection using two experimental systems. In the first, we used an anti-CD4 monoclonal antibody to deplete CD4+ T-cells in vivo in immunocompetent BALB/c mice. In the second system, we used mutant mice in which the gene encoding the CD4 antigen had been disrupted by homologous recombination, resulting in a lack of CD4+ T-cells. Challenge of either the anti-CD4 antibody depleted BALB/c mice or CD4 knockout mice with B. malayi infective-stage larvae demonstrated that mice lacking CD4+ T-lymphocytes were resistant to infection. These data indicate that CD4+ T-cells are not an obligate requirement for murine resistance to B. malayi.

Influence of gonadal steroids on susceptibility to Brugia malayi in scid mice.

In the present study, we demonstrate that male scid/scid mice have higher adult worm burdens than do female scid/scid mice following equal challenge doses of infective Brugia malayi L3 larvae. Gonadectomy of four week old immature mice has no effect on worm burden in either sex, suggesting that the gender dichotomy between males and females does not depend on continued presence of endogenous gonadal steroids. The worm yield from female, but not male, mice can be increased by prepubertal oophorectomy combined with administration of either estradiol or testosterone in depot form. Our results raise the possibility that prepubertal steroid pulse(s) result(s) in physiological changes in male scid/scid mice that enhance worm growth. These studies confirm earlier reports of epidemiological data in humans suggesting a sexual dimorphism in susceptibility to filarial infection. Our data suggest that this gender difference is not due simply to the presence of adult gonadal steroids, but rather to ontogenic differentiative actions of sex steroids in the host.

CD8+ T lymphocytes are not required for murine resistance to human filarial parasites.

Mice are resistant to the establishment of infection with the nematode parasite Brugia malayi, an etiologic agent of human lymphatic filariasis. We have recently shown that T and B lymphocyte-deficient C.B.-17 scid/scid mice are permissive for infection with this parasite, whereas coisogenic C.B.-17+/+ mice are resistant. This observation suggests that T and B lymphocytes that comprise the antigen-specific immune system orchestrate murine resistance to B. malayi. In order to define the component of the antigen-specific immune response that is responsible for this resistance, we have tested the susceptibility of beta 2M-/- mice to infection with B. malayi L3 larvae. These mice are homozygous for insertional disruption of their B2m genes, which encode beta 2-microglobulin, the small subunit of the major histocompatibility (MHC) antigens. They do not express beta 2-microglobulin and, as a consequence, fail to express the class I major histocompatibility antigens, and they do not develop the CD8+ class I MHC-restricted cytotoxic T cell subset. We find that these mice are completely resistant to B. malayi, indicating that the CD8+ T lymphocyte subset is not an obligate requirement for murine resistance to human filarial parasites.

Survival of Onchocerca volvulus in nodules implanted in immunodeficient rodents.

Onchocerca volvulus is an obligate human parasite, and its study has been difficult due to an inability to maintain it outside the human host. We report the successful transplantation of onchocercomata containing living adult O. volvulus worms into immunodeficient C.B.-17.scid/scid (scid) mice or athymic rnu/rnu (nude) rats. Living, motile worms containing viable microfilariae were present in onchocercomata recovered from scid mice or nude rats for up to 20 wk, establishing a novel animal model for future investigation of O. volvulus.

The immunodeficient scid mouse as a model for human lymphatic filariasis.

The C.B.-17-scid/scid mouse (hereafter referred to as the scid mouse) is homozygous for a recessive mutation at a locus that influences the assembly of intact immunoglobulin and T cell receptor genes. Therefore, scid mice cannot generate functional B or T lymphocytes, are profoundly immunodeficient, and have been reported to be receptive to reconstitution with human immune cells. In the present study, we injected scid mice with infective larvae of the human filarial parasite Brugia malayi. Within 6-10 wk after subcutaneous injection of infective L3 larvae, both male and female worms were observed in various stages of development in 90% of the mice. In animals tested 8 weeks or more after infection, microfilariae were detected in the blood or peritoneal cavity of 52% of the mice examined. Adult worms were observed in the lymphatics of the infected scid mice, where their presence was associated with lymphangitis and lymphangiectasia. These results suggest that the scid mouse model of lymphatic filariasis may be important in investigation of the interaction of the murine, and possibly the human, immune system with the lymphatic filarial parasite.

Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.

Heterozygous mammalian cell lines normally express both parental alleles at most autosomal loci. However, mutants can be isolated that fail to express one of the alleles. Using a murine pre-B cell line that is heterozygous for several loci on chromosome 12, including one encoding the cell surface antigen Ly-18, we found that one of the two Ly-18 antigenic forms was lost at a rate of 1.5 x 10(-5) per cell per generation. Molecular analysis revealed that a genetic marker distal to Ly-18 became homozygous. Analysis of the genotype of the mutants at the rDNA cluster, located close to the centromere, strongly suggests that the mutants arose by mitotic recombination within this multicopy locus.

Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.

Individual cells of the Caenorhabditis elegans secretory-excretory system were ablated by laser microbeam in various larval stages. Effects on growth, molting, osmoregulation, fertility, longevity, and dauer larva formation were tested. Single-cell ablations did not prevent subsequent molting, but ablation of the pore cell or the duct cell resulted in the absence of the normal cuticular lining of the excretory duct following a molt. When the pore cell, duct cell, or excretory cell was ablated, the animals filled with fluid within 12-24 hr and died within a few days, producing very few progeny. Ablation of the excretory gland cell, on the other hand, had no obvious developmental or behavioral effects. Excretory activity was monitored in dauer larvae by observing pulsation of the excretory duct in conditions of differing osmolarity. The rate of pulsation was quite variable over time in conditions of low osmotic strength, but average five- to six-fold higher than that observed in buffered saline. These observations, combined with the effects of laser ablation, lead to the conclusion that one function of the excretory system is osmoregulation.

Fine structure of the Caenorhabditis elegans secretory-excretory system.

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.

Medical experimentation in the elderly.

Participation in human experimental research constitutes a major problem for the geriatric subject. Because there is a high incidence of noncontagious disease in the elderly, they are the group most useful for the study of new therapeutic agents or procedures. However, normal aging processes, often coupled with disease of the central nervous system, render elderly persons less able to comprehend the nature and risks of such studies. These factors permit easy exploitation of geriatric subjects in medical experimentation, with possible exposure to a significant risk of serious drug reactions and unnecessary hospitalization. Recent federal regulations have given "special protections" to children, prisoners, and the mentally infirm in experimental research, to guard against abuse of their human rights. A basic requirement is that informed consent be carefully obtained and documented. Such "special protections" should now be extended to geriatric subjects so that there will be no further exploitation in the course of valid clinical research.